MORPHOLOGICAL VARIABILITY OF EUSTOMA HYBRIDS OBTAINED FROM INTERSPECIFIC CROSSES BETWEEN Eustoma grandiflorum X Eustoma exaltatum

1. Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, A.C., Carretera Sierra Papacal-Chuburna Puerto Km. 5.5, 97302, Mérida, Yucatán, México. 2. Campo Experimental Chetumal, INIFAP, Carretera Chetumal-Bacalar, Km 25, 77900, Quintana Roo, México. 3. Universidad Autónoma de Yucatán, Facultad de Ingeniería Química, Periférico norte, Km. 33.5, Tablaje catastral 13615, Chuburná de Hidalgo Inn, 97203, Mérida, Yucatán, México. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 30 November 2019 Final Accepted: 31 December 2019 Published: January 2020

The objective of this study was to determine the variability between hybrid lines of E. exaltatum x E. grandiflorum generated by Barba-González et al. (2017). Plants that were morphologically characterized were grown under ex vitro culture conditions in a tropical climate in Yucatan, Mexico.

Materials and Methods:ex vitro culture:
The seeds of the hybrids of Eustoma were provided by the Center for Research and Assistance in Technology and Design of the State Jalisco (CIATEJ). Hybrid plants were obtained from in vitro germination of hybrid seeds according to the protocol of Falcon Bautista et al., 2018. In vitro plants of four month of age with 5-8 cm in length with four pairs of developed leaves were transferred from in vitro culture to ex vitro conditions, 4 individuals were selected for each of the hybridized lines.
In vitro seedling roots were rinsed with distilled water; subsequently, a rinse was carried out with fungal solution of Binolate® 1g L -1 . The seedlings were placed in forks with Agrolite and sterilized organic soil at a ratio of 1:1. During 15 days, the Eustoma plants were covered with transparent nylon bags and placed in vitro culture conditions with photoperiod of 16 h light and 8 h dark, at a temperature of 23°C; subsequently, the transparent bags were removed and the seedlings were taken to ex vitro conditions.
During acclimatization process until that fully were developed plants, temperature, relative humidity and luminosity were recorded. ex vitro culture had two flowering periods that were recorded, the first period was from September to December and the second period from March to July. The average temperature recorded was 32.5°C, with a maximum temperature of 44.5°C recorded on May and a minimum temperature of 23.7°C on December. The percentage of maximum humidity was on October with a value of 78.31% and the minimum on April with a value of 31.7%. The maximum luminosity (Lx) is changed during April with a value of 225.25 and a minimum value of 31.5 on December.

Morphological characterization:
Morphological characterization was carried out based on guide TG / 197/1 Guidelines for the execution of the examination of the distinction, homogeneity and stability of Eustoma (UPOV, 2002), established by International Union for Protection of New Varieties of Plants (UPOV).
The morphological characteristics measurement was carried out in the period from March to June 2019, during the second flowering. Following what was described by Eustoma's guide TG / 197/1 (UPOV, 2002), the leaf observations were examined on the third sheet from the top; flower and pedicel observations were examined on the second flower that opened and on its inner face; internode length was measured in the fourth internode from the top.
The color observations were made using the HunterLab MiniScan EZ colorimeter, which records data on the CIELab scale (L = luminosity, -a / + a = green to red scale, -b / + b blue to yellow scale), during the anthesis in 3 petals per flower, the results were adapted to the color guide of the Roya Horticultural Society (RHS) as marked by the guide TG / 197/1 (UPOV, 2002) of Eustoma.

Statistical analysis:
The statistical parameters (average, minimum, maximum values, standard deviation and coefficient of variation) obtained for 14 quantitative and 10 qualitative morphological characters evaluated in 12 hybrid lines ( Table 1).
Analysis of main components was carried out with the objective of representing hierarchical relationships between the hybrids. Subsequently, the PROJ module was used to generate a two-dimensional projection of PCA using the NTSYS PC 2.1 software (Rohlf et al., 2000). A similarity matrix was made using the Euclidean distance coefficient based on the projection of main components to perform a cluster analysis using the UPGMA method (Unweighted Pair Group Method using Arithmetic averages) for the grouping of taxonomic units, using the NTSYS software version 2.1 (Rohlf et al., 2000). The selection of outstanding individuals was made by grouping all the individuals that make up each hybrid line, considering the separate values of the complete siblings, to make the grouping the 875 FIRST v6 software was used (Clarke, 2006). Statistical analyzes were performed with Statgraphics® statistical software.

Results and Discussion:-
Assessment of morphological characters in hybrids of the genus Eustoma statistically significant differences were found between the hybrids in relation to the morphological traits, except for the variable SD and LSTE, where the data did not present any difference in relation to the hybrid. High values as plant length (PL) and nodal segments number (NN) were observed in 1512063, 1512095, 1512096, 1512098 hybrids contrary the low values were obtained in 1512070, 1512093, 1512029 hybrids ( Table 2) (Table 2).
Correlation analysis of used variables in characterization of Eustoma genus hybrids, 13 significative relationships were obtained, which were considered values with an "r value" greater than 0.7. It is important to mention some significative correlations with shape plant, for example relation of Nodes number (NN)-PL presented a positive r value=0.7321, It means that both variables tend to increase at the same time. The same situation can be observed in floral traits like PN-FT with r value = 0.9996, SN-Flower type (FT) with r value = -1.000 and SN-Petals Number   Analysis of principal component allowed identifying the best traits to define the phenotypic variation between the hybrid lines. The 73.70% of total variation in 16 traits was explained in the first four principal components (Table  3).  Projection of 12 hybrid lines of Eustoma genus from the two first principal components permits to concentrate three groups, while in 1512063, 1512070, 1512093, 1512098 hybrid lines were considered independent groups (Figure 1). Those groups were dispersing in diagram; they showed higher phenotypic variability between materials, similar as  Twelve Eustoma genus hybrids were grouped by UPGMA method using the Euclidean coefficient on the principal components space. The dendrogram showed the same groups observed in Figure 1. Two principal groups and subgroups were formed. Seven groups (from AI to AIV and from BI to BIII) in a distance of 0.71 were observed.
The distribution related to ornamental traits for cut-flower was observed in the UPGMA dendrogram obtained from morphologic data. The group B integrated in its most by hybrids with higher PL, and Higher values in FN and NN. The group B was integrated by hybrids with intermediate values and low characteristics of PL, FN and NN. The flower color trait did not influence in grouping, which to suggest that this morphologic trait is not apt for grouping of Eustoma genus, endogamic lines of Viola wittrockiana also has been reported by Vemmos (2005). Grouping of Figure 3, 4 and 5, showed materials of a same hybrid in different categories. This variation between complete siblings came from heterozygosity of existing parental that to generate higher diversity inside of progeny.

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In conclusion, accord to traits of agronomic importance for ornamental market, in this study was selected the 1512063, 1512096, 1512098, 1512100 as outstanding hybrids.

Conclusion:-
Evaluation and analysis of morphologic traits helped to confirm the presence of outstanding hybrids, considering important agronomic traits to obtain cut-flower materials. The outstanding hybrids obtained were 1512063, 1512096, 1512098, and 1512100. Our studies reveled that outstanding hybrids obtained by interspecific crosses of E. grandiflorum and E. exaltatum could be cultured at high temperature.