Microsoft Word - 20-Agra_41748 222 Bioscience Journal Original Article Biosci. J., Uberlândia, v. 35, n. 1, p. 222-235, Jan./Feb. 2019 http://dx.doi.org/10.14393/BJ-v35n1a2019-41748 MORPHOLOGICAL AND STOMATAL CHARACTERIZATION OF Heliconia chartacea VAR. SEXY PINK INDUCED POLYPLOIDY CARACTERIZAÇÃO MORFOLÓGICA E ESTOMÁTICA DE Heliconia chartacea VAR. SEXY PINK INDUZIDAS A POLIPLOIDIA Marcelo Domingues Martins RAIZER1; Regina Caetano QUISEN2; Mágno Sávio Ferreira VALENTE1; Ricardo LOPES3; Maria Teresa Gomes LOPES1* 1. Faculdade de Ciências Agrárias, UFAM, Manaus, AM, Brazil; 2. Embrapa Florestas, Colombo, PR, Brazil; 3. Embrapa Amazônia Ocidental, Manaus, AM, Brazil. *mtglopes@hotmail.com ABSTRACT: The growth of the tropical flower market has demanded a consistent search for new varieties, primarily those endowed with an exotic profile, but that are also beautiful and durable. The genus Heliconia, naturally found in the Amazon region, is among the most prominent of tropical flowers. Looking to augment the genetic variability available in Heliconia chartacea var. Sexy Pink, biotechnological research was conducted with the application of colchicine to induce polyploidy in plants from this species. With that in mind, this study was undertaken to evaluate the establishment of plants in the field drawn from in vitro polyploidy induction assay and to determine the morphological and physiological characteristics of 38 H. chartacea var. Sexy Pink clones. The characterization analyzes were performed through 49 morphological descriptors and a stomatal density evaluation using microscopy. The genotype 35 exhibited the greatest morphological variations, with alterations in the position and coloring of the inflorescence, in addition to having the edges of the entire limbus. Genotype 18 featured the lowest amounts for plant height and inflorescence size, showing promise for research geared towards use in reduced environments. Some genotypes did not have any flowering and are recommended exclusively for landscape composition such as foliage, since their exotic characteristics allow for this. The genotypes that were evaluated displayed stomata with tetracytic morphology and guard cells that had no significant changes. However, genotypes with greater equatorial diameter and stomatal density were obtained in relation to the mother-plant. Overall, the induction of polyploidy allowed for clones to be obtained with a high variability for the characteristics of the leaf, pseudostem and inflorescence, with various attributes that confer a more efficient post-harvest management to some genotypes, in addition to favorable aspects for commercialized purposes as a cut flower. KEYWORDS: Floriculture. Heliconiaceae. Colchicine. Morphological descriptors. Stomatal density. INTRODUCTION The floriculture industry has one of the highest growth rates in national and international markets (JUNQUEIRA; PEETZ, 2014). The world floriculture production is growing at a rate of 10 per cent per year (HARISHA, 2017). Over the last few years, tropical flowers have been receiving particular attention due to the features that favor their scale production, such as beauty, exoticism, colors, shapes, resistance to transport and post- harvest durability (CARVALHO et al., 2012; PRIA, 2017). Yet, consumers seeking the latest innovations have forced plant producers and breeders to increasingly release new varieties to keep up with the demand in this market. In this regard, genetic breeding programs have been presented as a tool for diversifying and improving existing plant materials, particularly through using methods that increase the available genetic variability. The artificial induction of polyploidy has been an important method for creating new varieties of plant species, and this area has been gaining ground in the floriculture industry with the increasingly efficient use of mutagenic agents. One of the most widely used chemical agents is colchicine. Many varieties boasting "sales-friendly" features have been developed through their use. For example, there are plants with characteristics that are resistant to environmental adversities, like in Ziziphus mauritiana, which is resistant to water stress (NTULI; ZOBOLO, 2008), as well as new flower phenotypes and ornamental plants, such as Oncidium flexuosum (VICHIATO et al., 2007), Dendrobium nobile (UNEMOTO et al., 2009) and Heliconia bihail (CAVALCANTE FILHO, 2011). An increase in the chromosome number is usually illustrated through a higher cellular content, which can be reflected in an growth in the size of the plant organs and in a shift of its morphologies (WU et al., 2010). Thus, the association between the Received: 16/04/18 Accepted: 05/10/18 223 Morphological and stomatal... RAIZER, M. D. M. et al. Biosci. J., Uberlândia, v. 35, n. 1, p. 222-235, Jan./Feb. 2019 level of ploidy and morphological characteristics aids in identifying polyploid plants. The morphological characterization still allows the almost immediate adoption of plants with phenotypes that are pertinent to the producers and breeding programs, or even for differentiating and identifying potential genotypes that are to be designated as varieties. Because it is an easy and simple methodology, stomatal analysis has also been used for identifying supposed polyploids through the counting and comparative measurement of stomata (GŁOWACKA et al., 2010). Various polyploidy induction activities in vegetables used this characteristic to select polyploid candidates, like in Centella asiática (KAENSAKSIRI et al., 2011) and Lagerstroemia indica (WANG; LEI, 2012). Taking into account that part of the technologies applied in Brazil's flower production comes from other countries, research for tropical ornamental plants is still scarce, particularly with native species like Amazonian heliconias. These plants have an enormous potential for growth in the floriculture market, but the characterization of exotic germplasms and information on crop and post-harvest management deserves greater attention, even for more valuable plants like Heliconia chartacea var. Sexy Pink. Due to the need for ongoing research activities applied to the tropical flower and ornamental plants industry, Embrapa Amazônia Occidental coordinated a research project in 2009 titled “The development of technologies for heliconia production: a new niche market for the Amazon”. The purpose of this project was to define criteria for the production of Heliconia chartacea var. Sexy Pink in the environmental conditions surrounding the city of Manaus in the State of Amazonas that involved agronomic and biotechnological studies. One of the main products it sought to generate was establishing a population of polyploid plants in a field that demonstrated early interesting and innovative characteristics for this species. Consequently, this project sought to characterize Heliconia chartacea var. Sexy Pink plants that are obtained through induced polyploidization for the morphological and physiological characteristics that are of commercial interest. Therefore, this study was undertaken to evaluate the establishment of plants in the field drawn from in vitro polyploidy induction assay and to determine the morphological and physiological characteristics of 38 H. chartacea var. Sexy Pink clones. MATERIAL AND METHODS The genotypes used in the this study are linked to the collection of Heliconia chartacea var. Sexy Pink established in Embrapa Amazônia Ocidental's Experimental Field Headquarters in Manaus, Amazonas. These plants are derived from an induction of an in vitro polyploidy assay which consisted of the immersion of stem apices from a single mother-plant at different concentrations of colchicine (ranging from 0.01 to 0.10%). After the mutation induction, the explants were cultured and multiplied in MS medium (MURASHIG; SKOOG, 1962) supplemented with 0.15 mg of AIA (indole-3-acetic acid) and 3 mg of BAB (6-benzylaminopurine) (RAIZER et al., 2017), in vitro rooted (QUISEN et al., 2013), followed by acclimatization in a greenhouse and planted in a field in March 2011. In May 2014, the collection with 38 genotypes was transferred to a new area divided by clumps and established with a spacing of 3.0m between rows and 1.5m between plants, arranged in random blocks with four replications per genotype. Fertilization and cultivation cleaning followed Embrapa’s pre-established schedule and was based on recommendations for the cultivation of other tropical flowers (LUZ et al., 2005; SOUSA et al., 2009). Monthly evaluations were conducted of morphological descriptors in the genotypes established in the field for two consecutive years, and the criteria used was based on forty seven qualitative descriptors and two quantitative descriptors (Table 1) (CASTRO et al., 2007; LOGES et al., 2007). In order to have a more accurate identification of hue-related descriptors, a Munsell color chart was used. Visual assessments were made of the pseudo stem, leaves and inflorescences and the measurements were done with the aid of a digital caliper and tape measure in four plants from each genotype. To visualize the differences among H. chartacea genotypes based on the qualitative and quantitative descriptors, a dendrogram was designed through an analysis of clusters of the UPGMA type (Unweighted Pair Group Method using Arithmetical Averages) with the help of the statistical program R (R DEVELOPMENT CORE TEAM, 2017). The dissimilarity matrix was obtained using the Gower algorithm (1971) and the suitability of the cluster analysis to the original data was evaluated by the coefficient of co-optic correlation. 224 Morphological and stomatal... RAIZER, M. D. M. et al. Biosci. J., Uberlândia, v. 35, n. 1, p. 222-235, Jan./Feb. 2019 Table 1. Morphological descriptors used in the evaluation of induced polyploidy genotypes in H. chartacea var. Sexy Pink. Descriptors Identification of Phenotype Pseudo stem Plant height (“ALP”) Small-sized (˂ 1.50 m); Medium-sized (1.51-2.50 m); Large-sized (˃ 2.51 m) Diameter of pseudostem (“DPC”) Obtained a height of 30 cm from the soil Color of pseudostem (“CPC”) Greenish-yellow (2.5 GY 8/12); Light green (2.5 GY (8/8); Medium green (5GY 6/6); Dark green (7.5 GY 4/4) Waxiness of the pseudostem (“CER”) Absent; Small; Average; Much Hairiness of pseudostem (“PIL”) Absent; Small; Average; Much Intensity of anthocyanin (IAN) Intense; Average; Weak; Absent Leaf Length of third leaf (“CFO”) Measure from the base to the top of the Limbus Width of third leaf (“LFO”) Narrow (up to 10 cm); Median (of 20 to 30 cm); Large (more than 31 cm) Position of the leaves (“PFO”) Upright; Drooping; Arched Limbus (LIM) Torn; Not torn Green color of the underside of the leaf (“CFI”) Greenish-yellow (2.5 GY 8/12); Light green (2.5 GY (8/8); Medium green (5GY 6/6); Dark green (7.5 GY 4/4) Green color of the upper surface of the leaf (“CFS”) Greenish-yellow (2.5 GY 8/12); Light green (2.5 GY (8/8); Medium green (5GY 6/6); Dark green (7.5 GY 4/4) Shade of color on the underside of the main vein(“TCP”) Greenish-yellow (2.5 GY 8/12); Light green (2.5 GY (8/8); Medium green (5GY 6/6); Dark green (7.5 GY 4/4) Color of the side of the petiole (“CMP”) Purple (5R 3/10); Pink (2.5 R 7/2); Green (5GY 6/6); Brown (5YR 3/4); Transparent Waxiness of the limbus on the dorsal surface of the leaf (“CLD”) Absent; Small; Average; Much Waxiness of the limbus on the ventral surface of the leaf (CLV) Absent; Small; Average; Much Presence of spots on the limbus of the sapling (“PML”) No spots; Few spots; Scarce spots Shape of the limbus base (“FBL”) Both round; One round/ one tapered; Both tapered Comparison of the sizes of the limbus base (“CBL”) Equal; Unequal; Variable in the same plant Shape of the petiole base (“FBP”) Open with winged edges; Open with upright edges; Narrow with upright edges; Twisted edges on the inside part Edge with scaly base (“MBE”) Absent; Small; Average; Much Shape of the petiole edge (“FMP”) Closed; Semi-closed; Upright; Wide open Internal color of the sheaths (“CIB”) Purple (5R 3/10); Reddish-Pink (2.5 R 7/2); Green (5GY 6/6); Brown (5YR 3/4); Transparent Inflorescence Positioning of the inflorescence in relation to the stem (“PIH”) Upright; Drooping Length of inflorescence (“CI”) Short (10 cm ˂); Average (between 11 to 40 cm); Large (˃ 41 cm) Length of the 2nd bract (“CB”) Small (10 cm ˂); Average (between 11 to 40 cm); Large (˃ 41 cm) Width of the second bract (“LB”) Narrow (up to 10 cm); Median (of 20 to 30 cm); Large (more than 31 cm) Angle of the 1st bract in relation to the peduncle (“ABP”) ˃ 90°; 75°-90°; 45°-60°; 15°-30°; 0° Twisting the spine (“TR”) Present; Absent Visualization of the spine (“VR”) Visible; Covered by bracts 225 Morphological and stomatal... RAIZER, M. D. M. et al. Biosci. J., Uberlândia, v. 35, n. 1, p. 222-235, Jan./Feb. 2019 Width of the inflorescence (“LI”) Narrow (10 cm ˂); Average (between 11 to 40 cm); Large (˃ 41 cm) Leaf at the tip (1st bract) Present ; Variable; Variable in the same plant Shape of bract apex (“FAB”) Long (bulging); Narrow; Tappered Waxiness of the bracts (“CEB”) Absent; Small; Average; Much Hairiness of the bracts (“PB”) Absent; Small; Average; Much Symmetry of the bract (“SB”) Actinomorphic; Zigomorphic; Asymmetric Number of bracts in mature inflorescence (“NBI”) Small (5 ˂); Average (between 6 to 20); Much (˃ 21) Color of the spine (“CRA”) Dark Pink (5R 4/6); Light Pink (5R 4/4); Pink Orange (10R 7/8); Baby pink (2.5R 8/4); Pink Baby Orange (2.5R 7/8); Dark Red (5R 3/10); Medium Red (5R 4/10). Color of the external bract (“CBE”) Dark Pink (5R 4/6); Light Pink (5R 4/4); Pink Orange (10R 7/8); Baby pink (2.5R 8/4); Pink Baby Orange (2.5R 7/8); Dark Red (5R 3/10); Medium Red (5R 4/10). Color of the internal bract (“CBI”) Dark Pink (5R 4/6); Light Pink (5R 4/4); Pink Orange (10R 7/8); Baby pink (2.5R 8/4); Pink Baby Orange (2.5R 7/8); Dark Red (5R 3/10); Medium Red (5R 4/10). Color of the perigonium (CBP) White; Cream; Yellow (5Y 8/12) Color of the perigonium lobes (CLP) White; Cream; Yellow (5Y 8/12) Anthocyanin presence in the perigonium (PAP) Absent; In the basal part; Presence of stripes; Uniform coloring Presence of pollen (“PPO”) Absent; Small; Average; Much Color of the sepal (“CSE”) Greenish-yellow (2.5 GY 8/12); Light green (2.5 GY (8/8); Medium green (5GY 6/6); Dark green (7.5 GY 4/4) Petal Color (“CPE”) Greenish-yellow (2.5 GY 8/12); Light green (2.5 GY (8/8); Medium green (5GY 6/6); Dark green (7.5 GY 4/4) Ovary: Color of the unripe fruit (“OCI”) White; Cream; Yellow (5Y 8/12) Ovary: Color of the ripe fruit (“COM”) White; Cream; Purplish blue Flowering period (“PF”) Short (˂ 5 months); Medium (between 5 to 8 months); Long (˃ 8 months) To verify the existence of differences in the structures of a leaf blade, a clump of genotype with a good phytosanitary quality was selected, and three fully expanded leaves (third leaf) were collected. From the mid-part of each leaf, using a circular hole-puncher, a sample was taken of foliar tissue with 1 cm diameter. It was later placed in a petri dish and immersed in commercial hypochlorite solution until full depigmentation (24-48 hours). Next, using a soft-bristle brush, the abaxial and adaxial portions of all samples were separated, they were also stained with 1% toluidine blue and the blades were assembled with the addition of one drop of Canada balsam (Canada turpentine). The size of the stomas and stomatal density (DE - number of stomatas per unit area) of both parts of the leaf, abaxial and adaxial, were analyzed under an optical microscope and measured under a 40X lens, observing fifteen fields of view for each sample. Based on a micrometer scale for the eyepiece used, the stomatal density was determined through the ratio between the number of stomata and the area of the real field visible from the 40X lens (0.14 mm2). Slides were prepared with toluidine blue in order to evaluate the polar diameter (PD) and equatorial diameter (QD) of the stomata. They were observed in a clear chamber, in accordance with the Labouriau technique (CASTRO et al., 2009). The stomatal functionality ("FUN") was calculated from this data, given by the ratio between the polar diameter and equatorial diameter (CASTRO et al., 2009). The averages obtained for all variables were compared through the Scott Knott test at 5% probability. RESULTS AND DISCUSSION The clustering analysis based on morphological descriptors and Gower algorithm allowed for three distinct clusters to form (Table 1). Group 2 gathered most of the genotypes, while genotype 35 was the only one remaining alone in group 1, thus demonstrating that it had morphological characters distinct from the others. 226 Morphological and stomatal... RAIZER, M. D. M. et al. Biosci. J., Uberlândia, v. 35, n. 1, p. 222-235, Jan./Feb. 2019 These differences could be observed in the coloring of the inflorescence in the genotypes present in distinct groups (Figure 2). Figure 1. An analysis of the UPGMA cluster, based on qualitative and quantitative morphological characters and Gower algorithm, in H. chartacea var. Sexy Pink genotypes. Cophenetic correlation coefficient: r = 0.8552. The dotted horizontal line represents the cut estimated by the Mojema method. Figure 2. Inflorescences of H. chartacea var. Sexy Pink resulting from an in vitro polyploidy induction assay by somatic doubling using colchicine. (A) 29 Genotype; (B) 32 Genotype; (C) 35 Genotype. There was no presence of hairiness in the pseudo stem in any of the genotypes. The presence of hairs is an important characteristic in the production of heliconia, especially an excess, because it has a negative effect on post-harvest handling and transport that requires additional cleaning, because the presence of hairs leads to the retention of debris (CASTRO et al., 2007). The plant's height was over 1.5m in all genotypes (Table 2), which could hamper the handling and preparation of flower stems during post-harvest, because large stems are more prone to toppling or being ruptured. However, this is a characteristic that has already been described for this variety and the values obtained for the length of the floral stems are within what the market requires (ABBOTT et al., 2010). Clone 18 displayed the lowest plant height along with reduced inflorescences, which can be better used in landscaping projects that have a floristic composition in reduced environments. For the diameter of the pseudo stem (DPS), there was a significant variation among the genotypes, ranging from 23.32 (genotype 5) to 58.22mm (genotype 26). According to Farias et al. (2013), the DPS warrants attention due to its influence on flower resistance, handling, selection, packaging and post-harvest durability. A study conducted by Castro et al. (2007) with 30 heliconia genotypes, classified H. chartacea flower stems as large, indicating a high degree of difficulty in handling, and requiring special care for packaging and transportation. Both the diameter and stem length features are of highly important for the flower's resistance to wind, as stems that have larger diameters are also more rigid. According to Albuquerque et al. (2010), this is due to the carbon reserve contained in the stem that is used to prolong the potential longevity of the flowers. In this regard, Hermans et al. (2006) and Castro et al. (2007) claim that the longer the rod length and diameter, the greater the post-harvest durability. One explanation for the variation in the diameter of the stems is that it is due to the chromosome duplication in the evaluated genotypes. 227 Morphological and stomatal... RAIZER, M. D. M. et al. Biosci. J., Uberlândia, v. 35, n. 1, p. 222-235, Jan./Feb. 2019 Table 2. Characterization of H. chartacea genotypes when considering morphological descriptors of pseudo stem analysis. Gen. ALP1 DPC CPC CER IAN Gen. ALP DPC CPC CER IAN 1 Medium 34.44 Dark Green Much Weak 23 Large 30.00 Dark Green Much Weak 2 Medium 36.25 Mid Green Much Aus. 24 Medium 28.50 Light green Much Aus. 3 Medium 36.00 Mid Green Much Aus. 25 Medium 26.08 Mid Green Much Average 4 Medium 35.00 Mid Green Much Aus. 26 Medium 58.22 Mid Green Much Average 5 Medium 23.32 Mid Green Much Aus. 27 Large 26.50 Mid Green Much Aus. 6 Medium 25.99 Dark Green Much Weak 28 Medium 27.25 Light green Much Aus. 7 Medium 27.25 Mid Green Much Weak 29 Large 33.00 Mid Green Much Weak 9 Medium 29.32 Mid Green Much Weak 30 Medium 30.00 Mid Green Much Aus. 10 Medium 25.75 Light green Much Aus. 31 Medium 29.25 Mid Green Much Average 11 Medium 26.22 Mid Green Much Aus. 32 Large 26.50 Light green Much Average 12 Medium 29.50 Mid Green Much Average 33 Large 29.50 Mid Green Much Weak 13 Medium 38.22 Mid Green Much Weak 34 Medium 26.50 Mid Green Much Aus. 14 Large 27.52 Mid Green Average Weak 35 Medium 28.00 Mid Green Few Weak 15 Medium 26.52 Mid Green Much Weak 36 Medium 27.08 Dark Green Much Weak 17 Medium 26.72 Mid Green Much Weak 37 Medium 23.78 Mid Green Much Weak 18 Medium 26.72 Mid Green Much Weak 38 Medium 28.25 Mid Green Much Aus. 20 Medium 26.00 Mid Green Much Weak 39 Large 28.50 Dark Green Much Weak 21 Medium 27.25 Mid Green Much Aus. 40 Medium 26.08 Mid Green Much Weak 22 Medium 28.99 Mid Green Much Weak 41 Large 35.16 Dark Green Much Weak 1ALP = Plant Height; DPC = Diameter of the pseudo stem; CPC = Color of pseudo stem; CER = Waxiness of the pseudo stem; IAN = Intensity of anthocyanin. The presence of large amount of waxiness was constant in most of the evaluated genotypes. One noted exception was genotype 35, which featured waxiness in the younger pseudo stems and, as they developed, lost this characteristic. Another attractive feature of genotype 35 was the change in the leaf limbus (Table 3). Table 3. Characterization of H. chartacea genotypes for leaf morphological descriptors. Gen. CFO LFO LIM CFI CFS TCN CMP CLD CLV FBL CBL CIB 1 70.87 Median Torn Dark Dark Light green Purple Aus. Aus. Rounded Variable Light green 2 79.50 Median Torn Dark Dark Greenish-yellow Green Aus. Aus. Rounded Uneven Greenish-yellow 3 83.75 Median Torn Medium Medium Greenish-yellow Green Aus. Aus. Rounded Uneven Greenish-yellow 4 80.50 Median Torn Medium Dark Greenish-yellow Green Aus. Aus. Rounded Uneven Greenish-yellow 5 76.10 Median Torn Medium Dark Greenish-yellow Green Aus. Aus. Rounded Variable Light green 6 73.10 Wide Torn Dark Dark Greenish-yellow Purple Aus. Aus. Rounded Variable Light green 7 79.75 Median Torn Dark Dark Greenish-yellow Purple Aus. Aus. Rounded Variable Light green 9 67.77 Median Torn Medium Medium Greenish-yellow Purple Aus. Aus. Rounded Variable Light green 10 85.75 Median Torn Medium Medium Greenish-yellow Green Aus. Aus. Rounded Uneven Greenish-yellow 11 82.69 Median Torn Medium Medium Greenish-yellow Purple Aus. Aus. Rounded Variable Light green 12 85.00 Median Torn Dark Dark Greenish-yellow Purple Aus. Aus. Rounded Variable Light green 13 65.74 Median Torn Medium Medium Greenish-yellow Purple Aus. Aus. Rounded Variable Light green 14 67.82 Median Torn Medium Medium Light green Purple Aus. Aus. Rounded Variable Greenish-yellow 15 85.82 Median Torn Medium Medium Light green Purple Aus. Aus. Rounded Variable Light green 17 87.24 Median Torn Medium Medium Light green Purple Aus. Aus. Rounded Variable Light green 18 82.24 Median Torn Medium Medium Light green Purple Aus. Aus. Rounded Variable Light green 20 71.00 Median Torn Medium Medium Light green Purple Aus. Aus. Rounded Variable Light green 21 80.50 Median Torn Dark Dark Greenish-yellow Purple Aus. Aus. Rounded Variable Light green 22 80.43 Median Torn Medium Dark Greenish-yellow Purple Aus. Aus. Rounded Variable Light green 228 Morphological and stomatal... RAIZER, M. D. M. et al. Biosci. J., Uberlândia, v. 35, n. 1, p. 222-235, Jan./Feb. 2019 23 79.25 Median Torn Dark Light Greenish-yellow Purple Aus. Aus. Rounded Uneven Light green 24 81.00 Median Torn Medium Medium Light green Green Aus. Aus. Rounded Uneven Greenish-yellow 25 76.56 Median Torn Medium Medium Light green Purple Aus. Aus. Rounded Variable Light green 26 73.36 Median Torn Medium Medium Light green Purple Aus. Aus. Rounded Variable Light green 27 84.50 Median Torn Dark Dark Greenish-yellow Purple Aus. Aus. Rounded Uneven Light green 28 87.75 Median Torn Medium Medium Greenish-yellow Green Aus. Aus. Rounded Uneven Light green 29 85.25 Median Torn Dark Dark Greenish-yellow Green Aus. Aus. Rounded Uneven Light green 30 83.50 Median Torn Dark Dark Greenish-yellow Green Aus. Aus. Rounded Uneven Light green 31 93.00 Median Torn Dark Dark Light green Purple Aus. Aus. Rounded Variable Light green 32 87.00 Median Torn Dark Dark Light green Purple Aus. Aus. Rounded Uneven Greenish-yellow 33 91.00 Median Torn Medium Medium Light green Purple Aus. Aus. Rounded Uneven Light green 34 94.50 Median Torn Medium Medium Greenish-yellow Green Aus. Aus. Rounded Uneven Light green 35 83.00 Median Non-torn Medium Dark Light green Purple Average Average Pointy Variable Mid Green 36 88.56 Median Torn Medium Medium Light green Purple Aus Aus Rounded Variable Light green 37 61.37 Median Torn Medium Medium Light green Purple Aus. Aus. Rounded Variable Light green 38 80.25 Median Torn Dark Dark Greenish-yellow Purple Aus. Aus. Rounded Variable Mid Green 39 78.25 Median Torn Medium Medium Greenish-yellow Purple Aus. Aus. Rounded Uneven Light green 40 57.56 Median Torn Medium Medium Greenish-yellow Purple Aus. Aus. Rounded Variable Light green 41 83.73 Median Torn Dark Dark Mid Green Pink Aus. Aus. Rounded Uneven Light green CFO = Length of third leaf; LFO = Width of third leaf; LIM = Limbus; CFI = Green color of the underside of the leaf; CFS = Green color of the upper face off the leaf; TCN = Color shade of the underside of the main vein; CMP = Color of the edge of the petiole; CLD = Waxiness of the limbus on the dorsal surface of the leaf; CLV = Waxiness of the limbus on the ventral surface of the leaf; FBL = Shape of the limbus base; CBL = Comparison of the sizes of the limbus base; CIB = Inner color of the sheaths. It was the only one that exhibited the edges of the entire limbus and had more vivid coloring, characteristics that increase the possibilities of using this genotype in landscaping, as it becomes something that is more visually attractive (Figure 3). The presence of wax on the upper and lower surfaces of the leaf blade was also observed exclusively in the individuals of this genotype, which gives them extra protection against water loss from transpiration. Figure 3. Changes in leaf limb of H. chartacea var. Sexy Pink resulting from an in vitro polyploidy induction assay by somatic doubling using colchicine. (A) Genotype 12, (B) Genotype 35. Among the descriptors corresponding to the leaf area (Table 3), there was no variation for the leaf position, presence of spots on the limbus of the sapling, shape of the petiole base, edge of the base and the shape of the petiole edge. In this case, all genotypes exhibited arched, non-spotted leaves with a narrow petiole with upright and closed edges and lacking scariosum base. Assessments of the length of the third leaf (CFO) ranged from 57.56 (genotype 40) to 94.50cm (genotype 34) and only genotype 6 displayed leaves with a width greater than 30cm. By and large, the leaf descriptors presented great variability, however, a series of characterizations are needed to standardize these for the producer and, as a result, the end consumer because parameters such as beauty, exoticism and ideal coloring are subjective. Data shown in Table 4 again shows that the genotype 35 was detached from the others in relation to the position of the inflorescence. H. chartacea var. Sexy Pink normally exhibits drooping inflorescences and the genotype 35 produced erect and more compact inflorescences that are less than 40 cm in length which, according 229 Morphological and stomatal... RAIZER, M. D. M. et al. Biosci. J., Uberlândia, v. 35, n. 1, p. 222-235, Jan./Feb. 2019 to Albuquerque et. al. (2010) would classify this genotype as “suitable” for commercialization as a cut flower. The other genotypes would require individual packaging according to their size, which would require careful handling to avoid the rupturing the axis where the bracts are inserted. These genotypes also presented twisting of the spine, which makes the post-harvest preparation process excessively slow, including the use of protection for the bracts. All these resources include an additional component to the sale price. Table 4. Characterization of H. chartacea genotypes for morphological descriptors of inflorescence. Ge n. PIH CI TR LI FE CEB CRA CBE CBI CSE CPE OCI PF 1 Drooping Large Pres. Wide Variable Much Light pink Dark Pink Baby pink Light green Light green Cream Long 2 Drooping Large Pres. Wide Variable Much Dark Pink Light pink Baby pink Mid Green Mid Green Cream Long 3 Drooping Large Pres. Wide Variable Much Dark Pink Light pink Baby pink Mid Green Mid Green Cream Long 4 Drooping Large Pres. Wide Variable Much Dark Pink Light pink Baby pink Mid Green Mid Green Cream Long 5 Drooping Large Pres. Wide Variable Much Dark Pink Light pink Baby pink Mid Green Mid Green Cream Long 6 Drooping Large Pres. Wide Variable Much Dark Pink Light pink Baby pink Dark Green Dark Green Cream Long 7 Drooping Large Pres. Wide Variable Much Dark Pink Light pink Orange pink Mid Green Mid Green Cream Long 9 Drooping Large Pres. Wide Variable Much Dark Pink Light pink Baby pink Mid Green Mid Green Cream Long 10 Drooping Large Pres. Wide Variable Much Light pink Dark Pink Baby pink Light green Light green Cream Long 11 Drooping Large Pres. Wide Variable Much Light pink Dark Pink Baby pink Light green Light green Cream Long 12 Drooping Large Pres. Wide Variable Much Light pink Dark Pink Baby pink Light green Light green Cream Medium 13 NID NID NID NID NID NID NID NID NID NID NID NID NID 14 Drooping Large Pres. Wide Variable Few Orange pink Light pink Baby pink Mid Green Mid Green Cream Long 15 Drooping Large Pres. Wide Variable Much Dark Pink Light pink Baby pink Mid Green Mid Green Cream Long 17 Drooping Large Pres. Wide Variable Much Light pink Dark Pink Baby pink Light green Light green Cream Long 18 Drooping Avera ge Pres. Wide Variable Much Dark Pink Light pink Baby pink Mid Green Mid Green Cream Long 20 NID NID NID NID NID NID NID NID NID NID NID NID NID 21 Drooping Large Pres. Wide Variable Much Dark Pink Orange pink Baby pink Mid Green Mid Green Cream Long 22 Drooping Large Pres. Wide Variable Much Light pink Baby pink Baby pink Light green Light green Cream Long 23 Drooping Large Pres. Wide Variable Few Dark Pink Light pink Light pink Light green Light green Cream Long 24 Drooping Large Pres. Wide Variable Much Orange pink Orange pink Light pink Mid Green Mid Green Cream Long 25 Drooping Large Pres. Wide Variable Much Dark Pink Light pink Baby pink Mid Green Mid Green Cream Long 26 Drooping Large Pres. Avera ge Variable Much Dark Pink Light pink Light pink Light green Light green Cream Long 27 Drooping Large Pres. Wide Variable Much Light pink Light pink Light pink Mid Green Mid Green Cream Long 28 Drooping Large Pres. Wide Variable Much Dark Pink Baby pink Baby pink Mid Green Mid Green Cream Long 29 Drooping Large Pres. Wide Variable Avera ge Dark Pink Light pink Baby pink Mid Green Mid Green Cream Long 30 Drooping Large Pres. Wide Variable Few Dark Pink Orange pink Baby pink Mid Green Mid Green Cream Long 31 Drooping Large Pres. Wide Variable Much Dark Pink Orange pink Baby pink Mid Green Mid Green Cream Long 32 Drooping Large Pres. Wide Variable Much Dark Pink Orange pink Baby pink Greenish- yellow Greenish- yellow Cream Long 33 Drooping Large Pres. Wide Variable Few Dark Pink Light pink Baby pink Light green Mid Green Am. Long 34 Drooping Large Pres. Wide Variable Few Dark Pink Light pink Baby pink Dark Green Dark Green Cream Long 35 Upright. Avera ge Aus. Avera ge Pres. Few Red Red Red Greenish- yellow Greenish- yellow Cream Long 230 Morphological and stomatal... RAIZER, M. D. M. et al. Biosci. J., Uberlândia, v. 35, n. 1, p. 222-235, Jan./Feb. 2019 36 NID NID NID NID NID NID NID NID NID NID NID NID NID 37 NID NID NID NID NID NID NID NID NID NID NID NID NID 38 Drooping Large Pres. Wide Variable Much Dark Pink Orange pink Baby pink Light green Light green Cream Long 39 Drooping Large Pres. Wide Variable Much Dark Pink Light pink Orange pink Light green Light green Cream Long 40 NID NID NID NID NID NID NID NID NID NID NID NID NID 41 Drooping Large Pres. Wide Variable Much Dark Pink Dark Pink Baby pink Dark Green Dark Green Cream Medium PIH = Position of inflorescence in relation to stem; CI = Length of the inflorescence ; TR = Twisting of the spine; LI = Width of the inflorescence; FE = Leaf at the tip (1st bract); CEB = Waxiness of the bracts; CRA = Color of the spine; CBE = External bract color; CBI = Color of the internal bract; CSE = Sepal Color; CPE = Petal Color; OCI = Ovary : Color of unripe fruit; PF = Flowering period. *NID = Unclassified – The genotypes did not output any flowering by the end of the evaluation. For the inflorescences descriptors, there was no polymorphism for the length of the 2nd bract (average), width of the second bract (narrow), angle of the 1st bract relative to the peduncle (>90°), visualization of the soine (visible), apex shape (asymmetric), number of bracts in ripe inflorescence (average), perigone base color (cream), perigone lobes color (orange-yellow), presence of anthocyanin in perigonium (absent), presence of pollen (absent), ripe fruit color (purplish blue). The length of the bract classified as medium (11 to 40 cm) is significant because it usually represents the most prominence in flower arrangements and, therefore, in customer appreciation. Because there were no changes in this characteristic, we can assert that there was no loss of one of the main commercial features in this species. The absence of hairiness in the bracts is a positive characteristic because the presence of hair interferes greatly with the handling, preparation and transport. The damage to the handling is due to the fact that it occurs to the formation of a protective layer in the bracts, which can minimize the benefits of the conditioning treatment done for remove the heat from the field and restore the turgescence of harvested flowers. The hair layer can also host small insects, insecticide residues and dust particles, leading to intense handling and preparation and resulting in increasing production costs. Another negative aspect of the presence of hairiness with regards to transport is that the friction of the inflorescences causes hair to fall out in some bracts, damaging the appearance of the inflorescence and compromising its quality. Additionally, Broschat and Donselman (1983) warn of the necessity of packages that remain dry for suitable transport, and the hariness allows the bracts to be stored in excessive humidity, and can be transitioned to the packaging. The period for outputting the first inflorescence varied greatly between the genotypes. Genotypes 12 and 41 exhibited inflorescence up to eight months after planting (seven and eight months, respectively), while the other genotypes showed a longer flowering period, flowering only after eight months of planting. Genotypes 13, 20, 36, 37 and 40 did not output inflorescences at the end of the two years of the evaluation period. Heliconias are cut tropical flowers and the floral stem is the end- product of commercial significance that is used in arranging and preparing bouquets. Hence, the largest number of flower stem represents a lower cost of production for the producer, along with higher competitiveness and greater profitability. According to Broschat and Donselman (1983), the neotropical species of heliconia produce terminal inflorescences after outputting 4 to 5 leaves, which was not noted in these genotypes. One use for plants that did not output inflorescences would be in constructing landscapes as foliage, since its exoticity enables this purpose. Stomata of the genotypes offered tetrapeptic morphology and guard cells without significant changes. However, several genotypes displayed greater equatorial diameter and stomatal density compared to the mother-plant (Figure 4 and Table 5). These differences in the size and number of stomata per area have been common characteristics featured on several plant materials submitted to polyploidization, and are used as basic characteristics in indentifying polyploid individuals in various studies (CAVALCANTE FILHO, 2011; GŁOWACKA et al., 2010; WANG; LEI, 2012). 231 Morphological and stomatal... RAIZER, M. D. M. et al. Biosci. J., Uberlândia, v. 35, n. 1, p. 222-235, Jan./Feb. 2019 Figure 4. Format of stomata and guard cells in genotypes of H. chartacea var. Sexy Pink, induced polyploidy, in the Abaxial (A and B) and Adaxial (C and D) part of the leaves, in the genotypes 14 and mother- plant respectively. Bar = 20 µ m. On average, the stomatal density (“SD”) was 94.73% higher in the abaxial part compared to the adaxial part (Table 5). The adaxial epidermis' SD may be less responsive to environmental variations due to radiation being directly incidental upon it (CASTRO et al., 2009) and transpiration occurs mainly through the epidermis of the abaxial face (STRECK, 2003). Table 5. Average estimates of stomatal density (DE), polar diameter of stomatal (DP), equatorial diameter of the stomatal (DQ) and stomatal functionality (FUN), evaluated in 38 H. chartacea var. Sexy Pink genotypes in different regions of the leaf. Leaf regions Genotypes Adaxial Abaxial DE DP DQ FUN DE DP DQ FUN 1 28.57 a1 21.78 a 10.24 b 2.13 a 249.52 d 17.20 b 9.35 b 1.95 a 2 11.27 d 20.84 a 11.34 a 1.85 b 260.79 c 19.73 a 10.54 a 1.88 a 3 18.73 b 20.30 b 11.93 a 1.72 b 264.13 c 17.45 b 9.19 b 1.90 a 4 10.00 d 18.66 b 10.31 b 1.83 b 268.41 c 16.84 b 8.91 b 1.89 a 5 9.43 e 21.59 a 12.21 a 1.77 b 287.30 b 20.09 a 11.43 a 1.76 b 6 10.95 d 19.09 b 10.73 b 1.78 b 239.37 d 17.22 b 8.86 b 1.96 a 7 1.43 f 22.73 a 11.38 a 2.00 a 150.79 g 19.35 a 10.21 a 1.90 a 9 16.67 c 19.56 b 10.66 b 1.84 b 299.05 b 16.23 b 7.93 b 2.06 a 10 20.16 b 20.50 a 10.23 b 2.01 a 385.87 a 18.78 a 10.89 a 1.73 b 11 15.56 c 19.94 b 11.29 a 1.77 b 289.37 b 19.08 a 10.75 a 1.78 b 12 16.03 c 19.47 b 10.01 b 1.95 a 365.56 a 17.13 b 8.77 b 1.95 a 13 11.11 d 20.56 a 10.72 b 1.94 a 159.05 g 19.18 a 9.34 b 2.07 a 14 31.90 a 20.54 a 10.40 b 1.98 a 358.57 a 18.73 a 9.91 b 1.89 a 15 6.67 e 19.09 b 8.90 b 2.19 a 207.46 e 17.85 b 8.87 b 2.01 a 17 14.13 d 19.69 b 11.52 a 1.71 b 276.03 c 16.56 b 8.92 b 1.86 a 18 7.14 e 20.09 b 10.62 b 1.90 a 258.10 c 18.26 b 9.73 b 1.89 a 20 17.94 b 20.78 a 12.63 a 1.65 b 268.57 c 19.90 a 11.54 a 1.72 b 21 11.75 d 19.79 b 9.82 b 2.02 a 287.46 b 15.37 b 7.32 b 2.11 a 22 7.62 e 22.18 a 12.38 a 1.80 b 260.63 c 17.57 b 10.98 a 1.62 b 23 11.59 d 21.20 a 12.45 a 1.70 b 218.57 e 19.85 a 11.98 a 1.66 b 24 18.73 b 20.98 a 10.88 b 1.93 a 309.68 b 19.08 a 10.85 a 1.76 b 25 7.94 e 21.30 a 11.28 a 1.89 a 237.78 d 18.03 b 10.66 a 1.69 b 26 18.89 b 21.09 a 11.00 b 1.92 a 242.38 d 16.03 b 8.27 b 1.94 a 27 14.76 c 18.19 b 10.43 b 1.76 b 224.29 e 17.82 b 9.08 b 1.98 a 28 17.94 b 21.17 a 11.88 a 1.80 b 275.24 c 19.62 a 11.07 a 1.77 b 29 11.07 d 20.77 a 11.30 a 1.86 b 244.13 d 18.82 a 9.57 b 1.97 a 30 16.35 c 21.53 a 11.85 a 1.82 b 269.05 c 18.84 a 10.97 a 1.72 b 31 19.37 b 21.79 a 11.83 a 1.84 b 264.60 c 19.80 a 11.19 a 1.79 b 32 18.57 b 21.29 a 11.30 a 1.88 a 251.43 d 18.59 a 10.33 a 1.82 b 33 15.87 c 21.78 a 10.62 b 2.06 a 273.33 c 18.89 a 8.99 b 2.10 a 34 8.25 e 21.10 a 11.73 a 1.80 b 307.46 b 19.87 a 12.20 a 1.63 b 232 Morphological and stomatal... RAIZER, M. D. M. et al. Biosci. J., Uberlândia, v. 35, n. 1, p. 222-235, Jan./Feb. 2019 35 0.79 f 20.75 a 12.74 a 1.64 b 183.97 f 20.97 a 12.48 a 1.68 b 36 11.59 d 21.28 a 12.59 a 1.70 b 196.35 f 21.24 a 12.31 a 1.73 b 38 13.65 d 22.17 a 11.75 a 1.89 a 258.89 c 20.24 a 11.09 a 1.83 b 39 9.84 d 23.62 a 11.72 a 2.02 a 270.95 c 17.80 b 8.37 b 2.13 a 40 19.84 b 20.23 b 11.40 a 1.78 b 246.35 d 20.16 a 11.65 a 1.73 b 41 11.59 d 18.85 b 10.25 b 1.88 a 209.05 e 17.70 b 8.46 b 2.09 a Mother-plant (control) 10.81 d 20.48 a 10.88 b 1.90 a 260.10 c 18.46 a 9.88 b 1.89 a Overall mean 13.88 20.71 11.20 1.86 259.99 18.54 10.08 1.86 CVe (%)2 14.87 6.34 9.23 8.5 6.56 6.33 9.89 8.4 1Averages followed by the same letter in each column pertain to the same group according to the Scott and Knott grouping criterion at 5% probability; 2Coefficient of experimental variation. The highest SD were observed in genotypes 1 and 14 for the adaxial part of the leaves, whereas genotypes 10, 12 and 14 were the ones with the highest values for the abaxial part. The lowest values were found in genotypes 7 and 35 for the adaxial part and in genotypes 7 and 13 for the abaxial part of the leaves, differences caused by the induced mutation. Castro et al. (2009) relate that a higher number of stomata per unit of leaf area may confer a high adaptability to environments that are dry or have a lack of available water. Therefore, a higher SD can enable a stomatal opening in a shorter period of time, permitting an adequate capture of CO2 and reducing the time in which these stomata become open. This reduces transpiration and allows for the plants to better adapt to conditions in which water is scarce (MIGLIORANZA; OLIVEIRA, 2013). A higher stomatal functionality (FUN) may be associated with reduced transpiration because the stomata becomes more elliptical (CASTRO et al., 2009; BATISTA et al., 2010), the reduced transpiration may also be associated with a greater SD, which is frequently observed under conditions with a greater amount of radiation and less available water (SOUZA et al., 2007; CASTRO et al., 2009). Therefore, among the varieties with the highest FUN and SD values, genotypes 1, 12 and 14 stand out, and the best averages were obtained by genotype 14, both for the adaxial as well as the abaxial part, showing that this genotype presents the most functional stomata. CONCLUSIONS The induction of polyploidy in H. Chartacea var. Sexy Pink allowed for clones to be obtained with a high variability for leaf characteristics, pseudostem and inflorescence, with various attributes that confer a more efficient postharvest management to some genotypes, in addition to favorable aspects for commercialized purposes as a cut flower. The presence of genotypes with changes in the position, coloring and size of the inflorescence, in addition to modifications in the leaf limb that left them with whole edges, showed promise for future projects geared towards the production of cut flowers and landscaping in H. chartacea var. Sexy Pink. The evaluated genotypes present stomata with tetracytic morphology and guard cells that do not have significant changes. However, some genotypes show a larger equatorial diameter and stomatal density in relation to the mother-plant. The next steps of this research are the analysis of the DNA content by flow cytometry for polyploidy identification of the genotypes. ACKNOWLEDGEMENTS The authors thank the National Council for Scientific and Technological Development (CNPq) and Amazonas Research Foundation (FAPEAM) for financial support. RESUMO: A expansão do mercado de flores tropicais tem demandado uma constante procura por novas variedades, principalmente aquelas dotadas de perfil exótico, mas ainda apresentando beleza e durabilidade. Dentre as flores tropicais de maior destaque, se encontram as do gênero Helicônia, sendo estas naturalmente encontradas na região Amazônica. Visando aumentar a variabilidade genética disponível em Heliconia chartacea var. Sexy Pink, pesquisas biotecnológicas foram realizadas com a aplicação de colchicina para indução a poliploidia em plantas da espécie. Deste modo, o objetivo do presente trabalho foi avaliar as plantas estabelecidas em campo, provenientes dos ensaios de indução à poliploidia in vitro para determinar as 233 Morphological and stomatal... RAIZER, M. D. M. et al. Biosci. J., Uberlândia, v. 35, n. 1, p. 222-235, Jan./Feb. 2019 características morfológicas e fisiológicas de 38 clones de H. chartacea var. Sexy Pink. As análises de caracterização foram realizadas por meio de 49 descritores morfológicos e avaliação da densidade estomática por microscopia. O genótipo 35 foi o que apresentou as maiores variações morfológicas, com alterações na posição e coloração da inflorescência, além de possuir as bordas do limbo foliar inteiras. O genótipo 18 apresentou os menores valores para altura da planta e tamanho das inflorescências, mostrando-se promissor para pesquisas voltadas ao uso em ambientes reduzidos. Alguns genótipos não tiveram floração, sendo recomendada a sua utilização exclusivamente para composição paisagística como folhagens, já que sua exoticidade permite esta finalidade. 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