Introduction

Poinsettia or “cuetlaxochit” (Euphorbia pulcherrima Willd. Ex Klotzsch) is the most important species of the Euphorbia genus, and one of the pot plant with higher economic impact, with annual sales of hundreds of millions of dollars (^{USDA, 2015}). In Mexico, its sale initiates during the second week of November and continues until December. The demand is focused on interior design, gardens and public spaces although its medicinal use is also reported (^{Colinas-León, 2009}). In the last decade, the surface destined to its cultivation has increased, which has been reflected in its economic aspect. Around 30 million poinsettia plants are produced per year, with a value of production of $522,713.03; the states of Morelos, Michoacán, Puebla, District Federal, Jalisco, Estado de México and Oaxaca (^{SIAP, 2017}) stand out, where around 30 varieties are cultivated.

Most of ornamental species produced in Mexico depends 100 % on improved imported varieties, as propagules or as seeds; this create a dependence on the exterior for vegetal material. In the case of E. pulcherrima, genetic breeding has been realized in United States of North America, France, Norway, Austria and Germany, where varieties have been generated, whose main characteristics are the mechanical resistance to cold, in addition to color, shape and size of the bracts, which are different from the existing varieties, size and vigor of the plant, reduction of the sensibility to ethylene, and higher duration of the bracts (^{Taylor et al., 2011}).

Imported varieties present problems as the reduction or the excess of vegetative growth, early beginning of flowering (in September) or lack of pigmentation in the bracts; the previous mentioned is a consequence of the climatic differences among production zones and the generation areas of the varieties. The previous said indicates that poinsettia varieties must be generated in each country where it is produced.

By being the original center of poinsettia (E. pulcherrima), Mexico relies on native plants and varieties named of sun (grow and flower in sun conditions in gardens), which have been promoted and rudimentary domesticated in home gardens, being considered close to the native species, both types of plants could be used as a source of genes to generate Mexican varieties (^{Villegas et al., 2015}).

Seeds are the base of the genetic diversity of the species and essential element in conventional genetic breeding. In the case of poinsettia species, seeds are generally produced by genetic breeders (^{Bernuetz, 2001}). ^{Canul-Ku et al. (2013)} reported that through hybridization they obtained nine genotypes whose female progenitors were plants of native E. pulcherrima from the states of Oaxaca, Morelos, Puebla and Guerrero, and as male progenitors they used Freedom Red and Prestige commercial varieties.

The efficient production of seeds depends on several factors, plant genotype, the number of repetitions of pollination, temperature and light of the environment. In poinsettia species, the production of seeds obtained by crossed pollination and auto-pollination, is generally low (^{Bernuetz, 2001}). The capacity of seed production in this species varies according to the varieties used as female and male progenitors (^{Rodríguez et al., 2016}). The higher the production of seeds, the higher is the probability to find new phenotypes; that is why the ideal is progenitors with higher capacity of developing fruits and seeds per fruit (one, two or three seeds).

The ploidy level of progenitors is a determining factor in seed production; ^{Bernuetz (2001)} reported that E. pulcherrima has 2n = 28 or 56 genetic information; the number of somatic chromosomes of the cultivars is 28 or 56, those with 2n = 28 are diploids, those that own 2n = 42 chromosomes are called triploids, but according to ^{Ewart & Walker (1960)} they could be hexaploids. According to ^{Milbocker & Sink (1969)}, hybridization between diploid cultivars generate an abundant production of seeds; however, when crossing female diploids with tetraploids, less than 1 % of viable seeds are obtained, the development of endosperm in most of the ovules do not progress over the initial stage of the cells, the previous mentioned suggesting that endosperm may be the determining factor in the establishment of the seed in the diploid x tetraploid cross.

E. pulcherrima fruits are tricarpelar schizocarps with a central column and one ovule per carpel, in which a maximum of three seeds are formed, when not all of them are formed, the mature fruit is narrower (^{Tokuoka & Tobe, 2003}), these are structures that present explosive dehiscence releasing seeds up to 2 m of distance. ^{Rodríguez et al. (2016)} indicate that the absence of seeds generate an abscission of fruits. ^{Canul et al. (2012)} reported that poinsettia seeds collected in the states of Nayarit, Morelos and Guerrero presented polar diameters of 7.98; 7.56, and 7.17 mm, equatorial diameters of 7.04; 6.21 and 6.08 mm, and biomass of 174, 131, and 120 mg respectively. ^{Vargas (2012)} observed that E. pulcherrima sun plants produce seeds without human intervention, because flower pollination is entomophile. Nevertheless, for genetic breeding, the pollination has to be controlled and the efficiency of fecundation depends on the selected progenitors for crosses, as pointed out by ^{Canul et al. (2012)}; ^{Huang and Chu (2008)} indicated that progenitors have to produce high quantity of viable pollen, its period of releasing has to be extended, and they must have the capacity to produce a high quantity of seeds.

As previously mentioned, E. pulchherima seeds are used for genetic breeding, each seed can generate a different phenotype, undesired situation when the purpose is to produce homogenous plants for commercialization, in this case propagation is done through cutting (^{Rangel-Estrada et al., 2015}) or stakes (^{Colinas et al., 2015}).

The above mentioned indicates that it is necessary to generate knowledge allowing to support genetic breeding programs in E. pulcherrima, with the purpose to obtain national varieties adapted to environmental conditions of the country, with competitive characteristics with imported varieties and at available prices for producers. Based on that, the objective of the investigation was to evaluate the production of fruits and seeds in crosses of two poinsettia sun varieties with four shade, to determine which progenitors are more convenient to use in a genetic breeding program.

Material and Methods

The research work was performed from July 2016 to June 2017, in the Experimental Field of the Faculty of Agricultural Sciences of the Autonomous University of the State of Morelos, in Cuernavaca, Morelos, Mexico, with geographical location of 18°58’54,71’’ N and 99°13´59,14’’ W, and 1876 masl. The climate is semi-warm semi-humid A(C) w2, presents a rainy season with annual average precipitations of 1200 mm and an annual average temperature of 21.5 °C (^{García, 1981}).

Used sun varieties (E. pulcherrima for garden) were Amanecer navideño and Juan Pablo, whose characteristics are: resistance to sun, narrow bracts (5 cm approx.), early beginning of flowering (final of October for the State of Morelos, México), and duration of the flowering of 80 days approx.; these varieties flower without the need to cover them with black plastic to give them additional darkness hours to those of the time of cultivation, as it has to be done with imported shade varieties.

Shade varieties (from greenhouse) were: Festival red, of compact plants with 8 to 9 branches, withstand direct sunlight without showing damages, shiny red color of bracts, medium beginning of flowering (in Morelos it begins the second week of November), duration of the flowering of 100 days approximatively, 2) Ice punch, which had irregular-shaped plants with 7 to 8 branches, wide leaves (6 to 8 cm), webbed-shaped, late beginning of flowering (the fourth week of November, in Morelos) and duration of flowering of 100 days approx., the color of the bracts is a combination of red with white (Red group-c 43, yellow group-d10); 3) Premium Marble, regular-shaped plants with 6 to 7 branches, cream and pink color of bracts, wide leaves and bracts with entire leaf margin, intermediate beginning of flowering; 4) Cortez burgundy, is of compact plants with 7 to 8 branches, wide bracts (6.54 cm in average), intermediate beginning of flowering (in Morelos it begins the second week of November), duration of flowering of 120 days, purple bracts (Purple gray group N77). Regarding genetic characteristics, sun E. pulcherrima are diploid as well as Festival red and Premium marble, while Ice punch is a tetraploid variety (^{Milbocker & Sink, 1969}).

Progenitor plants of sun varieties were grown outdoor in 10’ pots using substrate made of 70 % of compost and 30 % of “tepojal” (small pumice stone of volcanic origin wrapped with clay, lighter than tezontle); shade varieties were maintained under plastic greenhouse conditions (with minimum temperature from 12.2 to 15.7 °C, maximum from 25.2 to 27.0 °C, relative humidity from 51 to 73 %). Nutrition was realized with highly soluble fertilizer 20-20-20 every 15 days; fungicides were applied in a preventive way by using Carbendazim (1 g·L^-1), N-trichloromethyl dicarboxymide (2 mg·L^-1), Mancozeb (1 g·L^-1), Iprodion (1 g·L^-1) and Metalaxyl-M + Chlorothalonil (2 mg·L^-1) products, in total five applications of fungicides were applied; Abamectin insecticide (1 mL·L^-1) were applied for mite control.

To realize crosses, in varieties that serve as female progenitor, 15 hermaphrodite flowers were selected per repetition; these flowers were emasculated and anthers were eliminated with fine forceps before anthesis, as they were appearing. The cross was realized when the stigma was receptive (Figure 1a) by depositing pollen of the variety that serves as male progenitor; pollinized flowers (Figure 1b) were covered with glassine bags.

Figure 1 Receptive stigma of poinsettia (A), Pollinized hermaphrodite flower (B), Growing fruit (C). 

Eight treatments (intercrosses) were evaluated, derived of the combination of two sun varieties (Amanecer navideño and Juan Pablo) with four shade varieties (Festival red, Ice punch, Premium marble and Cortez burgundy), a Completely Randomized Experimental Design was used with 10 repetitions per cross. Variables of characteristics of fruits and seeds were evaluated.

Fruits were harvested when they were dry and fruit set was registered, the number of formed fruits by repetition, and the percentage regarding the number of total crosses per repetitions was calculated. Moreover, in each fruit, length and diameter was evaluated with a Truper® digital Vernier, and its biomass in an Ohaus® analytic balance. Posteriorly, the seed of the fruits of each cross was harvested and deposited in plastic tubes identified with the number of fruits, the repetition and the origin of the cross, the number of seeds per fruit was assessed.

Length and diameter were evaluated in seeds with a Truper® digital Vernier, as well as the biomass per seed in an Ohaus® analytical balance. The cross of Juan Pablo x Ice punch did not produce any fruit, therefore characteristics of fruits and seeds were not measured. Obtained data in the variables was analyzed by means of variance analysis (ANOVA) and Tukey Multiple Comparison Test (Tukey, p≤0.05) with SAS 9.0 (^{S.A.S, 2002}) software. Values of evaluated variables in percentage were arcsin transformed previously to analysis.

Results and Discussion

The variance analysis showed significant effect (p≤0.01) of the cross for the five assessed characteristics of the fruit and of seed (Table 1), indicating that female and male progenitors involved in the intercrosses affect the evaluated variables.

Regarding fruit set, Amanecer navideño generated fruits when it was crossed with the four male progenitors, with an average of fruit set of 31.64 %. In the case of Juan Pablo variety, the fruit set was achieved only in three of the four performed crosses, since as observed (Figure 2), it did not develop any fruit when it was crossed with Ice punch. However, the female progenitor gave the highest average percentage of developed fruits (36.72 %). Festival red and Premium marble generated similar results as male progenitors (63.99 and 64.63 % of fruit set, respectively); in contrast, Ice punch and Cortez burgundy generated a few fruits and even Ice punch did not generate fruits when crossing it with Juan Pablo (Figure 2). The best crosses for obtaining fruits were Juan Pablo x Premium marble and Amanecer navideño x Festival red.

Figure 2 Set of fruit (%) in crosses of two sun varieties of poinsettias with four varieties of shade. An: Amanecer navideño, Jp: Juan Pablo, Fr: Festival red, Ip: Ice punch, Pm: Premium marble, Cbur: Cortez burgundy. MSD: 18.14. Each datum is the average of ten repetitions. 

The low percentage of fruit set obtained with Ice punch and Cortez burgundy might be due to the fact that pollen viability of Cortez burgundy was the lowest of the four shade varieties (^{Vargas, 2012}). Another reason may be what indicated by ^{Milbocker & Sink (1969)}, who reported that crossing of diploid poinsettia with tetraploid generate lesser that 1 % of viable seeds, suggesting that endosperm can be the determining factor for growth and development of seeds in crossing of diploid x tetraploid; ^{Petit et al. (1997)} pointed out that triploid seeds can be unviability of endosperm. The above mentioned results in the fact that the seed cannot develop and the fruit fall weeks after pollination, as highlighted by ^{Wang et al. (2005)}, who indicated that unviability of hybrid embryo in crossing of diploid x tetraploid and the absence of seeds result in the fall of fruits.

Fruit set of the cross between Amanecer navideño x Festival red was similar to the one reported by ^{Canul-Ku et al. (2015)} who obtained 68 % of developed fruits. Nevertheless, these results were overtaken by 83.6 % obtained in cross of Juan Pablo x Premium marble. It is important to highlight that handling during pollination also affect fruit set, since in intercrossing realized by ^{Rodríguez et al. (2016)}, a maximum of 24 % of fruit set was obtained when crossing Amanecer navideño x Festival red and in this case 68.98 % of fruit set was obtained. In addition to choose receptive hermaphrodite flowers, anthers with fresh pollen have to be chosen, released the same day of the pollination.

The length of the fruit varied from 14.29 to 15.44 mm, with an average of 15.07 mm. Fruits with the highest length were those produced by crosses of Juan Pablo x Festival red, Premium marble x Cortez burgundy, as well as those of Amanecer navideño x Festival red; similarly, fruits of higher diameter correspond to those produced by Juan Pablo x Premium marble and by Cortez burgundy. Regarding biomass, fruits of higher weight were those of the crosses of Juan Pablo with Festival red and Premium marble, as well as those of Amanecer navideño by Festival red. Fruits of lower weight correspond to the crosses of Amanecer navideño x Premium marble and by Cortez burgundy (Table 2).

One of the most important aspects for this study and which affect characteristics of the fruit is the quantity of seeds that were able to develop. ^{Tokuoka & Tobe (2003)} described that they are tricarpelar schizocarps with a central column and one ovule per carpel. It was observed that a maximum of three seeds were formed and when all are not formed, the fruit is narrower and of lower weight (Figure 1c). Rinehart (pers. comm. 1997), cited by ^{Bernuetz (2001)}, indicated that the triocular ovary of E. pulcherrima can contain a maximum of three seeds, although an average of two per ovary is generally obtained.

In this study, it was observed that in four of the seven crosses, fruits with more than two seeds were produced, corresponding to crosses where Festival red and Premium marble participated as male parents, therefore being the best male parents; on the contrary, fruits with less seeds were those generated by both female parents with Cortez burgundy (Table 2). The above mentioned is related to pollen quantity and viability, since Cortez burgundy had pollen with less viability. The formation of seeds was also affected by the level of ploidy of parents; E. pulcherrima plants from sun varieties that were diploid and crossed with Festival red and Premium marble generated a higher quantity of seeds; otherwise, being crossed with Ice punch and Cortez burgundy (tetraploid varieties), less seeds were formed (^{Milbocker and Sink, 1969}). The quantity of seeds per fruit was higher than what ^{Rodríguez et al. (2016)} obtained, whose obtained 1.1 seeds per fruit, this may be due to a better knowledge of poinsettia flower phenology.

It is desirable that fruits have two or three seeds, but it is also necessary that they are big enough to contain a higher quantity of endosperm to use during germination process. Seeds of higher length were those produced by fruits of Juan Pablo variety, although also those of intercrossing between Amanecer navideño x Ice punch; in four of the seven crosses, 7 cm length (approx.) seeds were obtained (Table 3), similar to what reported by ^{Canul et al. (2012)} who observed an average of 7.5 mm for poinsettia seeds of different origins; the smallest seeds (5.8 mm) were those of crossing between Amanecer navideño x Cortez burgundy, similarly to the results obtained by ^{Rodríguez et al. (2016)}.

The diameter of the seeds varied from 5.0 to 5.6 mm, most of the crosses generated seeds with 5.0 to 5.1 mm. This value was lesser than 6.3 mm reported by ^{Canul et al. (2012)} and ^{Rodríguez et al. (2016)}. Seeds of Amanecer navideño x Ice punch and those of Juan Pablo x Cortez burgundy has correspondence of higher diameter and higher length (Table 3).

Biomass is the best indicator of seed quality, because they can have high length and diameter but they can be “vain” in higher and lower degree. Most of the seeds of crossing had an average biomass of 107 to 120 mg, there was correspondence between seed size and weight only in seeds of Juan Pablo x Cortez burgundy which were the biggest and with the highest biomass. These results were lower than those reported by ^{Canul et al. (2012)} for poinsettia seeds collected in the states of Nayarit, Morelos and Guerrero (0.174; 0.131 and 0.120 g, respectively); they were also lower to those reported by ^{Rodríguez et al. (2016)} who had seeds from 0.221 to 0.395 g. The obtained results are attributed to the presence of more than two seeds by fruit and a higher quantity of fruits per plant, causing a lower biomass.

Conclusions

The research allows to determine that parents involved in the controlled crossings of E. pulcherrima have a determining effect on fruit set and on the quantity and quality of produced seeds. Amanecer navideño variety was found to be the best variety to be used as female parent in comparison with Juan Pablo, since it generated seeds when it was crossed with the four varieties used as male progenitors. Similarly, results indicated that shade varieties Premium marble and Festival red were the best male progenitors for producing a higher fruit set and seeds.