Impaginato 283 Adv. Hort. Sci., 2022 36(4): 283­292 DOI: 10.36253/ahsc­12981 Biological effects of some Colchicum autumnale L. extracts on tissue devel­ opment of two varieties of Ocimum basilicum L. I.­C. Moroșan (*), L.C. Ivănescu, M. Mihășan, Ș.M. Olaru, M.­M. Zamfirache Alexandru Ioan Cuza University of Iași, Bulevardul Carol I, 11, 700506 Iași, Romania. Key words: Colchicine, basil, bulbs, flowers, meadow saffron. Abstract: Colchicum autumnale L. is a perennial herb from the Colchicaceae family with an unusual life cycle, and it is characterized by an underground corm and hypogynous flowers that appear in autumn. Its medicinal importance is represented by its primary alkaloid, colchicine, which has been studied for its anti­inflammatory and antimitotic properties and used in the treatment of some diseases and artificial polyploidy induction in plants. This study aims to determine and evaluate the biological effects induced by treatment with C. autumnale extracts on tissue development in test plants, represented by two Ocimum basilicum L. varieties: ‘Italiano Classico’ and ‘Aromat de Buzău’. Morpho­anatomical observations and measurements and photosynthetic pig­ ments analyses were employed. Results show unusual shapes of leaves, differ­ ences in stomata size and density, and heteromorphic cells in leaves and epi­ cotyl’s structure in both studied varieties of basil treated with C. autumnale extracts. 1. Introduction Colchicum autumnale L., generally known as meadow saffron, naked lady, or wild saffron (Nagesh et al., 2011), is a colchicine­containing species widespread throughout Europe. It belongs to the Colchicaceae family, which comprises perennial herbs characterized by an underground corm and hypogynous flowers with six tepals (Bowles, 1924; Nordenstam, 1998, in Jung et al., 2011). The biochemical profile of this species reveals the presence of alkaloids, phenolics, terpenoids, glycosides, and other bioactive compounds (Davoodi et al., 2021; Hailu et al., 2021). The alka­ loid concentration in this species varies depending on the plant organ; it was reported that it varies in seeds (0.5­1.2%), fresh flowers (1.2­2%), fresh leaves (0.15­0.4%), and fresh bulbs (0.1­0.6%); among all alkaloids, colchicine constitutes 50­70% of the total alkaloid content, followed by some small amounts of colchicoside, demecolcine, and other tropolone derivatives (Kupper et al., 2010). These variations are due to the fact that (*) Corresponding author: morosan.ioana@gmail.com Citation: MOROȘAN I.­C., IVĂNESCU L.C., MIHĂȘAN M., OLARU Ș.M., ZAMFIRACHE M.­M., 2022 ­ Biological effects of some Colchicum autumnale L. extracts on tissue development of two varieties of Ocimum basilicum L. ­ Adv. Hort. Sci., 36(4): 283­ 292. Copyright: © 2022 Moroșan I.­C., Ivănescu L.C., Mihășan M., Olaru Ș.M., Zamfirache M.­M. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) 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 relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 5 April 2022 Accepted for publication 17 October 2022 AHS Advances in Horticultural Science https://doi.org/10.36253/ahsc-12981 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2022 36(4): 283­292 284 secondary metabolites biosynthesis and accumula­ tion are influenced by genetic, morphogenetic, and environmental factors (Yang et al., 2018). Despite its toxicity, colchicine has been used in the treatment of gout, Familial Mediterranean Fever (FMF), and other diseases (Nagesh et al., 2011), and its antimitotic properties have been exploited in plant breeding for the production of polyploid crops (Roberts and Wink, 1998, in Jung et al., 2011) and ornamental species (Manzoor et al., 2019). To extract colchicine from the plant source, the most commonly used method is Soxhlet, and the best choice of solvent for this method has been reported to be methanol for C. autumnale and other species of the Colchicaceae family (Finnie and Van Staden, 1991; Pandey and Banik, 2012, in Çankaya et al., 2019). Colchicine binds to tubulin dimers and pre­ vents microtubule assembly, inducing microtubule depolymerization and preventing mitotic spindle for­ mation (Caperta et al., 2006). It was shown that colchicine treatment affects specific morphological (stomatal size), physiological (photosynthetic rate), and biochemical (chlorophyll content) indices in vari­ ous plant species (Cao et al., 2018; Trojak­Goluch et al., 2021), but the effects of colchicine derivatives or C. autumnale extracts in plants are unknown. This study aims to evaluate the effects of C. autumnale methanolic extracts on seed germination and plantlet tissue development by measuring sever­ al morpho­anatomical and photosynthetic indices to determine the allelopathic effect and the impact of the extracts of this plant species on other plants. For this purpose, Ocimum basilicum was chosen as a test species for its culinary, ornamental, medicinal, and economic importance and specific morpho­anatomi­ cal characteristics: four­edged stem (rectangular­ quadrangular shaped) with four ribs and ovate­lance­ olate opposite leaves with attenuate serrate edges, uniseriate pluricellular tector hairs, peltate (four­ celled head) and capitate (one or two­celled head) glandular hairs that appear on the surface of the stem and leaves (Zamfirache et al., 2008; Nassar et al., 2014), that make potential abnormalities easy to spot. 2. Materials and Methods Plant material The seeds of O. basilicum ‘Italiano Classico’ and ‘Aromat de Buzău’ were purchased from commercial sources (Unisem S.A., Iași, and S.C.D.L. Buzău). A total of 32 individual plants of C. autumnale were collect­ ed from a pasture in Voroneț, Suceava County, lat. 47.58889° N, long. 25.90861°E, alt. 576.68 m, in October 2019. The colchicine standard was provided by the Institute of Biological Research, Iași, Romania. The bulbs and flowers of C. autumnale were kept in an oven at 65°C for 12 hours to stop enzymatic reactions and then dried at room temperature (21±1°C) for 7 days, away from any source of light. Subsequently, the dry material was ground in an electric grinder, placed in glass jars wrapped in alu­ minum foil, and stored in the refrigerator until used in extract preparation. Extraction and quantification of colchicine content in extracts The extraction was processed in a Soxhlet appara­ tus in methanol (Sigma Aldrich, Germany), using 5 grams of powder from each organ (bulb and flower), according to the method of Franz and Koehler (1992, in Alali et al., 2004). The extraction of colchicine from the dried plant material was performed until the sol­ vent in the extraction chamber was clear, in the fol­ lowing way: 5­6 cycles (1­1.5 cycles/hour) for bulbs and 17­18 cycles (2­3 cycles/hour) for flowers. After extraction, the methanol was evaporated in a rotary evaporator (IKA RV3 Eco, Germany). Each dry extract was weighed and then dissolved in 50 ml of 70% methanol. Two colchicine­containing extracts were obtained, one from the bulbs and abbreviated BE (bulb extract) and one from flowers ­ abbreviated FE (flower extract). The quantification of the colchicine in the extracts was performed by RP­HPLC, according to Alali et al. (2004). The separation was performed on a Shimadzu Prominence HPLC system (2x LC20AD pumps, SIL20AC autosampler, CT20AC oven, SPD M20A DAD detector) using a Zorbax Eclipse XDB ­ C18 (250 mm length, 3 micron particle size) column, with acetoni­ trile as mobile phase A, and 3% acetic acid (Sigma Aldrich, Germany) as mobile phase B. Elution was performed at a flow rate of 1 ml/min using the fol­ lowing program: 0­3 min 90% B isocratic, 3­11 min 90­40% B gradient, 11­12 min 40% B isocratic, 12­13 min 40­90%B gradient, 13­20 min 90% B isocratic. A volume of 20µl of colchicine standard and 5 µl of each extract were injected. Colchicine was detected at 245 nm and eluted at 13.4±0.08 minutes. Chromatographic data were acquired using the Shimadzu LC solution Software and manually inter­ Moroșan et al. ‐ Colchicum autumnale extracts effects on plant tissue development 285 preted. For calibration, a range of 0.2 to 5 µg colchicine was used. Experimental design The two extracts were used in treatments, pure or diluted with distilled water (1:1) on the seeds and on the cauline apexes of O. basilicum ‘Italiano Classico’ and ‘Aromat de Buzău’ potted plantlets (Fig. 1). Three controls (C0 = distilled H2O, C1 = 35% MeOH, C2 = 70% MeOH) were prepared according to the methanol concentration in extracts and dilutions. The method of treatment consisted of applying extracts on O. basilicum seeds distributed in Petri dishes. The seeds were previously sterilized with 3% H2O2 (5 minutes) and 5% NaClO (5 minutes). The treatment was applied by soaking the filter paper with 2 ml of pure or diluted extracts (20 seeds/Petri dish x 3 replicates for each variant of treatment). The seeds were kept in a thermostat at a temperature of 20±1°C and a 17h/7h light­dark cycle per day for 14 days and were watered once every two days. The experiment was performed in triplicate. Final Germination Percentage (FGP) was calculated according to the formula by Bezini et al. (2019): FGP = n/N × 100 where n represents the number of germinated seeds at the end of the germination test and N is the total number of seeds. The other treatment application strategy was based on some methods used for polyploidy induc­ tion by colchicine treatment (Suzuki et al., 2005; Ye et al., 2010; Kushwah et al., 2018) and was carried out by applying the treatment on basil plantlets on day 18 after planting, by soaking cotton balls applied on the cauline apexes with 100 µl of extract per day for three consecutive days. The plantlets were kept in pots with Compo Sana soil (peat + perlite) at 21±2°C and 17 hours of light per day and were watered once every three days. The experiment lasted for 95 days (18 days of pre­cultivation and 75 days of monitoring of plant development) and the treated plantlets were analyzed at the end of the experiment (day 75 after treatment). Morphological observations of treated plantlets The macromorphological differences in leaves’ shape and stem development that were observed between variants of treated O. basilicum plantlets of both varieties were photographed. For micromorphology analysis, the plant material consisting of the first leaves was prepared in the fol­ lowing way: for each variant 2 first node leaves from 3 different plants were cut (1 cm2) and transferred in successive acetone baths, critical­point dried with CO2, and covered with a 10 nm gold (Au) layer using an EMS 550x sputter coater. Leaf samples of each surface were analyzed using scanning electron microscopy (Tescan Vega II SBH electron microscope from the Faculty of Biology, ‘’Alexandru Ioan Cuza” University of Iași, Romania) with VegaTC software. Stomata size (on both leaf surfaces) measurements were made using the ImageJ software for 5 stomata on 6 leaves. Stomata, tector, and glandular hairs den­ sities were determined by counting their occurrences on 1 mm2 of leaf surface on 6 leaves. Anatomy analysis of treated plantlets Epicotyl fragments from 3 plantlets of each treat­ ment were selected for sectioning and examination. The plant material, previously kept in 70% ethanol was sectioned using a hand microtome and botanical razor. The sections were stained through the double staining method (with ruthenium red and iodine green), then placed on slides, observed through an optical microscope (Euromex bScope BS.1153­Pli) using a 10x (0.25) lens, and photographed using Xiaomi Mi A1 camera (12 MP, f/2.2, 26 mm (wide), ½.9”, 1.25 µm, PDAF). Epicotyl circumference was measured using ImageJ and compared to control variants. Photosynthetic pigments content assay of treated plantlets The content of photosynthetic pigments in both O. basilicum varieties was analyzed according to Sumanta et al. (2014) using ethanol (Chemical Company S.A., Iași, Romania) as a solvent for leaf extracts (fresh leaves from 3 plants of each variant of treatment were weighted and milled with quartz sand, then dissolved in ethanol and filtered). The contents of chlorophyll a, chlorophyll b, and Fig. 1 ­ Treatment application strategies. Adv. Hort. Sci., 2022 36(4): 283­292 286 carotenoid pigments were calculated with formulas given in the reference article. The data was processed in GraphPad Prism version 9.3.0. Chlorophyll fluorescence measurement of treated plantlets For the measurement and evaluation of chloro­ phyll fluorescence (indirect measurement of photo­ chemical efficiency of photosystem II = ΦPSII and electron transport rate = ETR), 3 leaves were selected from different individuals from each treatment (from plantlets that were pre­exposed to dark conditions), and were analyzed using the Hansatech Ltd. PAM Fluorometer. The data was imported from the data system of the Hansatech device to Parview32 soft­ ware and analyzed in GraphPad Prism version 9.3.0. Statistical analyses Statistical calculations and comparations were performed in GraphPad Prism version 9.3.0., using Two­way ANOVA and Tukey’s multiple comparisons test for all morphological: stomata size (n= 3), stoma­ ta, tector and glandular hairs density (n= 3) on both leaf surfaces, anatomical: epicotyl circumference (n= 3), biochemical: chlorophyll a, b and carotenoids con­ tent (n= 3), and physiological indices: final germina­ tion percentage for treated seeds (n= 20) and photo­ system II efficiency and electron transport rate (n = 3) for treated plantlets. Values in graphs were repre­ sented as means ± SEM, and for Tukey’s multiple comparison tests, statistical significance is marked on graphs in the following way: **** = p<0.0001, *** = p<0.001, ** = p<0.01, * = p<0.05. Where no asterisk is present, results are not statistically significant. 3. Results Colchicine content quantification in extracts The detected colchicine (Fig. 2a) concentrations were 0.119±0.007 mg/ml colchicine in the bulb extract (Fig. 2b) and 0.286±0.015 mg/ml colchicine in the flower extract (Fig. 2c). Germination test The final germination percentage recorded on seeds treated with extracts revealed that the bulb extract and a concentration of 70% methanol impede the germination of all seeds, regardless of the variety of basil. O. basilicum ‘Italiano Classico’ seeds had a higher germination percentage than the ‘Aromat de Buzău’ cultivar under treatment, but the latter Fig. 2 ­ Chromatograms for colchicine standard (a) and for bulb (b) and flower (c) extracts. Moroșan et al. ‐ Colchicum autumnale extracts effects on plant tissue development 287 showed a better germination percentage than its control variant when treated with diluted flower extracts (Table 1). The inhibitory effects of treat­ ments (with extracts and methanol) effects were sta­ tistically significant (**** = p<0.0001) compared to C0. Effects of C. autumnale extracts were also signifi­ cant (p<0.0001), compared to their corresponding methanol control (1:1 BEt and 1:1 FEt to C1; BEt and FEt to C2). Other statistical comparisons are present­ ed in supplementary materials (SM) Tables S1­S6. Morphological observations of treated plantlets Plantlet morphology. In ‘Italiano Classico’ basil plantlets, the epicotyl’s growth was inhibited; the plantlets remained short (mainly when treated with bulb extracts), and their epicotyls had very short internodes (Fig. 3). The upper leaves were darker in color compared to the control and displayed unusual shapes: elongated or bifurcate, with straight or incomplete margins and diverted nervures (Fig. 4 c, d, e). Treated ‘Aromat de Buzău’ plantlets in different stages of growth and development, with various stem heights and ramifications are reported in figure 5. In some plantlets, the main epicotyl’s growth was either inhibited or necrotic, which strongly stimulat­ ed the ramifications, mostly when treated with pure Table 1 ­ Final germination percentage of O. basilicum seeds under treatment with C. autumnale extracts Fig. 3 ­ Morphology of O. basilicum ‘Italiano Classico’ plantlets treated with C. autumnale extracts. a) H2O Control; b) MeOH; c) diluted (1:1) bulb extract; d) pure bulb extract; e) diluted (1:1) flower extract; f) flower extract. Fig. 4 ­ Morphology of O. basilicum ‘Italiano Classico’ leaves from the basal node of plantlets treated with C. autum‐ nale extracts. a) H2O Control; b) MeOH; c) diluted bulb extract; d) diluted flower extract; e) pure flower extract; leaves from plantlets treated with pure bulb extract could not be photographed because of insufficient plant material. Fig. 5 ­ Morphology of O. basilicum ‘Aromat de Buzău’ plantlets treated with C. autumnale extracts. a) MeOH and H2O Control; b) dilu­ ted (1:1) bulb extract; c) pure bulb extract; d) diluted (1:1) flower extract; e) pure flower extract. BE= bulb extract; FE= flower extract; t= treatment; ****= p <0.0001. Test variant Final germination percentage (%) ‘Italiano Classico’ ‘Aromat de Buzău’ C0 (H2O) 80±1.92 68.89±7.78 C1 (MeOH 35%) 70±11.55 **** 23.33±4.41 **** 1:1 BEt 0±0 **** 0±0 **** 1:1 FEt 40±18.93 **** 33.33±6.01 **** C2 (MeOH 70%) 0±0 **** 0±0 **** BEt 0±0 **** 0±0 **** FEt 0±0 **** 0±0 **** 288 Adv. Hort. Sci., 2022 36(4): 283­292 flower extract. The upper leaves were much smaller than those from the control variant and displayed unusual shapes (Fig. 6c, d, e), with incomplete mar­ gins and deviated or bifurcated nervures (Fig. 6f). Leaf micromorphology observations of treated plantlets. A typical basil leaf (of a plant treated with H2O = C0) observed through scanning electron microscopy has diacytic and anomocytic stomata, glandular and tector hairs (tector hairs appeared only on the adaxial leaf surface), and epidermal cells with wavy sidewalls (Fig. 7a). The leaf epidermis of plantlets treated with methanol appears dehydrated and uneven (Fig. 7b), tector hair morphogenesis and elongation are stimulated, and glandular hairs with an altered shape of the glandular cells occur (Fig. 7c). Leaves from ‘Italiano Classico’ plantlets treated with flower extracts showed heteromorphic or twin stomata (Fig. 7d), accentuated corrugations of the epidermal cell sidewalls (Fig. 7e), and abnormalities of the glandular hairs’ shapes (Fig. 7f, g). Bulb extracts treatments only stimulated tector hairs mor­ phogenesis. The aspect of a typical leaf surface of ‘Aromat de Buzău’ basil plantlets is similar to ‘Italiano Classico’, but when treated with bulb extracts, epidermal cells appear elongated, the shape of the sidewalls is altered, and the morphogenesis and elongation of tector hairs occur following the treatment with methanolic flower extracts (Fig. 7h). Micromorphology measurements of treated plantlets. Stomata area and density. Stomata size sig­ nificantly increased on both leaf surfaces of ‘Italiano Classico’ plantlets treated with diluted bulb extract (p<0.05 when compared to C0, p<0.0001 when com­ pared to C1 on the inferior leaf surface, and p<0.0001 when compared to C0 and C1 on the superior leaf sur­ face) and pure flower extract (p<0.001 when com­ pared to C0, p<0.0001 when compared to C2 on the inferior leaf surface, and p<0.0001 when compared to C0 and C2 on the superior leaf surface) and only on the abaxial leaf surface when treated with diluted flower extract (p<0.0001 when compared to C0 and C1)(Fig. 8a), and their density increased in plantlets treated with pure bulb extract on the inferior leaf surface (p<0.01 when compared to C0 and p<0.05 when compared to C2) (Fig. 8c, d). The density of tec­ tor hairs increased significantly when plants were treated with 70% MeOH on the inferior leaf surface as a response to chemical stress. Stomata size increased on both leaf surfaces of ‘Aromat de Buzău’ basil plantlets treated with pure bulb extract (p<0.0001 when compared to C0 and C2) and only on the adaxial leaf surface when the plantlets were treated with diluted flower extract (p<0.0001 when compared to C0 and C1) (Fig. 8a, b). Stomata density increased on both leaf surfaces of Fig. 6 ­ Morphology of O. basilicum ‘Aromat de Buzău’ leaves from the basal node of plantlets treated with C. autum‐ nale extracts. a) H2O Control; b) MeOH; c) diluted bulb extract; d) pure bulb extract; e) diluted flower extract; f) pure flower extract. Fig. 7 ­ Abnormalities observed on O. basilicum leaf surfaces after treatment with methanol or C. autumnale extracts. a) typical aspect of an abaxial leaf surface; b) typical aspect of an adaxial leaf surface; c) glandular hair with an altered shape; d) twin stomata; e) epidermal cells with very wavy sidewalls; f) twin glandular hairs; g) glandular hair with a modified shape; h) tector hair morphogenesis and an elongated pluricellular tector hair. Moroșan et al. ‐ Colchicum autumnale extracts effects on plant tissue development 289 plantlets treated with pure flower extract (p<0.001 when compared to C0 and C2) (Fig. 8c, d). Glandular and tector hairs density. The density of tector hairs on the abaxial leaf surface increased in all ‘Italiano Classico’ basil plantlets, being highest in those treated with pure bulb extract and in the ‘Aromat de Buzău’ plantlets treated with flower extracts and 70% methanol, compared to C0, where they did not appear. The treatment with extracts did not significantly impact either basil variety on the adaxial leaf surface. On the other hand, glandular hairs density did not change on either leaf surface of ‘Italiano Classico’ basil plantlets, but it was signifi­ cantly higher on the adaxial leaf surface of ‘Aromat de Buzău’ plantlets treated with 70% methanol (SM ­ Fig. S1). Epicotyl anatomy of treated plantlets By examining the epicotyl anatomic structure of plantlets treated with C. autumnale extracts, some changes in the cross­section’s outline, an uneven lig­ nification of the xylem vessels, and heteromorphic cells in the cortical parenchyma and marrow, and an increase in the size and density of tector hairs were observed. The contour of the cross­section through the epi­ cotyl of a typical basil plantlet (control) is square, with 4 prominent ribs, which is a characteristic aspect of the species from the Lamiaceae family. The single­ layered epidermis has numerous single­celled, long tector hairs and glandular hairs. The layers below the epidermis are differentiated into two subzones: the angular collenchyma and the parenchymal cortex, with 4­6 layers of rounded cells that leave small air gaps between them. The central cylinder comprises conductive tissues in which elements of the primary structure (generated from procambium) and the sec­ ondary structure (generated from cambium) are observed. Secondary xylem and libriform (sclerenchy­ ma wood) fibers can be observed in approximately equal proportions. The marrow is parenchymal­meat­ ic (Fig. 9, 10). ‘Italiano Classico’ basil plantlets treated with dilut­ ed bulb extract showed a weaker lignification of the xylem bundles and an elongated, rectangular contour of the epicotyl cross­section. The asymmetry of the epicotyl’s shape in plantlets treated with flower extract is correlated with an increased number of cell layers on one side. Many cell layers indicate individu­ als with thicker epicotyls than the control. Also, more multicellular tector hairs were observed compared to the control (Fig. 9). ‘Aromat de Buzău’ basil plantlets treated with diluted bulb extract displayed a thinner epicotyl than the control variant, shorter tector hairs, and an aerif­ erous cavity in the pith, whereas the epicotyl of the plantlets treated with undiluted bulb extract dis­ played some heteromorphic cells in the parenchymal cortex, which led to an abnormal outline of the cross­ Fig. 8 ­ Average stomatal area on abaxial (a) and adaxial (b) leaf surfaces and density on abaxial (c) and adaxial (d) leaf surfaces of treated O. basilicum plantlets (BE = bulb extract; FE = flower extract; t = treatment; **** = p<0.0001; *** = p<0.001; ** = p<0.01; * = p<0.05). Fig. 9 ­ Epicotyl cross­section from O. basilicum ‘Italiano Classico’ plantlets, after treatment with C. autumnale diluted bulb (b), flower (c), and pure flower (d) extracts, compared to the control (a) (epicotyls of plantlets treated with pure bulb extract could not be analyzed because of insufficient plant material). Adv. Hort. Sci., 2022 36(4): 283­292 290 section. Similar modified characteristics were observed in ‘Aromat de Buzău’ basil plantlets treated with flower extract: when treated with a diluted flower extract, the epicotyl displayed an increase in the number of tector hairs, in contrast with undiluted flower extract treatment, where it decreased. In addition, a weak lignification of xylem vessels, het­ eromorphic parenchymal cells, and an overall outline thinning of the epicotyl were possible consequences of the treatment (Fig. 10). Epicotyl circumference increased in ‘Italiano Classico’ basil plantlets when treated with pure flower extract (p<0.01 when compared to C0 and p<0.0001 when compared to C2). The plantlets treat­ ed with the bulb extract could not be analyzed due to the fragility of the plant material and some anom­ alies of the epicotyl (very short internodes). In con­ trast, ‘Aromat de Buzău’ basil had a much lower epi­ cotyl circumference when treated with diluted bulb extract (p<0.0001 when compared to C1 but not sig­ nificant when compared to C0) but higher in plantlets treated with pure bulb extract (p<0.0001 when com­ pared to C0 and C2) (Fig. 11). Photosynthetic pigments content of treated plantlets The applied treatments did not affect chlorophyll a and carotenoid pigments content in both basil vari­ eties. Chlorophyll b content was highest in plantlets treated with 70% methanol (SM Fig. S2). Photosystem II efficiency and electron transport rate of treated plantlets The photosystem II efficiency of both varieties and the electron transport rate (ETR) of ‘Aromat de Buzău’ basil plantlets were not significantly affected by the applied treatment. In ‘Italiano Classico’ basil, the electron transport rate was very high when plantlets were treated with 70% MeOH (C2) (SM Fig. S3). 4. Discussion and Conclusions C. autumnale pure extracts inhibited the germina­ tion of seeds of both ‘Italiano Classico’ and ‘Aromat de Buzău’ basil varieties, probably because of the high concentration of methanol, but they could also have an inhibitory effect on germination and growth of weeds in plant cultures due to their allelopathic properties. More seeds of the ‘Italiano Classico’ basil variety germinated than ‘Aromat de Buzău’, but the latter withstood the treatment on cauline apex better than the Italian basil. Morphological observations showed that the treatments have similar effects on leaf morphogene­ sis of both basil varieties, at macroscopical and microscopical levels, with some differences in the response of a particular basil variety to flower or bulb extract treatments. The epidermis dehydration, alter­ ations in glandular hair shapes, and stimulated mor­ phogenesis and elongation of tector hairs could appear because of the solvent used in treatments Fig. 10 ­ Epicotyl cross­section from O. basilicum ‘Aromat de Buzău’ plantlets, after treatment with C. autumnale diluted bulb (b), flower (c), and pure bulb (d) and flower (e) extracts, compared to the control (a). Fig. 11 ­ Epicotyl circumference of O. basilicum plantlets after treatment with C. autumnale extracts. BE= bulb extract; FE= flower extract; t= treatment; **** = p<0.0001; ** = p<0.01; * = p<0.05; epicotyls of plan­ tlets treated with pure bulb extract could not be analy­ zed because of insufficient plant material. Moroșan et al. ‐ Colchicum autumnale extracts effects on plant tissue development 291 rather than due to the applied treatments with Colchicum autumnale extracts. The increase in the number of tector hairs observed on the epicotyl epi­ dermis, or their occurrence where they wouldn’t usu­ ally appear (on the abaxial leaf surface), might indi­ cate a defensive response to the chemical stress induced by methanol. In contrast, an increased densi­ ty of secretory hairs on the leaf adaxial surface of ‘Aromat de Buzău’ plantlets treated with methanol might indicate a better capacity to protect them­ selves, as glandular hairs’ main role is to protect the plant against external factors: herbivores and pathogens, extreme temperatures, excessive loss of water, and competing plants by secreting lipophilic substances that act as repellents or poisons (Hazzoumi et al., 2020). The observations on the anatomy of the plantlet epicotyl confirmed the infor­ mation from the literature that colchicine affects xylem differentiation and thickening because of the disappearance of wall microtubules (Pickett­Heaps, 1967), which might lead to abnormal morphogenesis of the mature plants’ stem that develops in a pla­ giotropic position. Another interesting observation is that heteromorphic cells induced by the treatment with C. autumnale extracts that appear in the leaf structure and in the epicotyl might indicate the pres­ ence of mixoploid tissues, which could also explain the abnormal leaf shapes and the epicotyl develop­ ment anomalies. The treatment with C. autumnale extracts did not affect biochemical and physiological indices of the photosynthetic apparatus of treated basil plantlets. Only treatment with methanol might influence pho­ tosynthesis due to its impact on chlorophyll b con­ tent and electron transport rate. 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