Genetic material

In this work, two populations of popcorn were used as females, one was Compuesto Amarillo (CA), which comes from a stabilized population formed from a hybrid of the Palomero Toluqueño race (Mex-5) × Yellow Pearl of the United States of America, from which yellow caryopses were selected and after a genetic recombination between them, the CA population originated.

The other population was the North American Yellow Pearl Popcorn (NAYPP) race, which corresponds to the type of corn most commonly grown commercially in the corn belt of the United States. The males used were the two previous materials, the inbred line CML-349, with semicrystalline endosperm, white caryopsis and early maturity adapted to highlands and a population of landrace corn of the Cacahuacintle (CAC) race of mealy endosperm, native to the Texcoco Valley and characteristic of the High Valleys of central Mexico.

Experimental sites

The pollination experiments were established during the spring-summer cycle of 2018 in two environments of the Central High Valleys, one was the locality of Montecillo, municipality of Texcoco, State of Mexico (19° 27’18” north latitude and 98° 54’ 26” west longitude, at an altitude of 2 240 m, with average annual temperature of 12-18 °C and average annual rainfall of 637 mm) (^{García, 1988}). The other environment was Santa María Zacatepec, municipality of Juan C. Bonilla, Puebla (19° 06’ 00” north latitude and 98° 20’ 00” west longitude, at an altitude of 2 206 m) (^{INEGI, 2009}).

Experimental design, experimental unit, and treatments

The combination of genotypes (2 females x 4 males) resulted in a total of eight treatments, which were established in both localities under an experimental design of randomized complete blocks with three repetitions. The experimental unit of the female plants consisted of plots of two furrows 5 m long and 0.8 m wide, where the sowing was at the rate of three seeds every 50 cm and after the second work, a thinning was carried out to leave two plants, having a final density of 55 000 plants ha^-1. Additionally, plots of male plants were established, which consisted of six furrows with the same sowing specifications described; pollen was taken from these plots to form the different treatments.

To avoid the presence of pollen from other sources on female plants, the baby corns of these were covered with number 8 glassine paper bags before exposure of stigmas; also, prior to anthesis, the tassels of male plants were covered with tassel bags, made of 12-gauge kraft paper, 30 cm long and 17.5 cm wide, during the early hours of the morning. The collection of pollen from the covered tassels was carried out after 10 in the morning and was deposited on the baby corns with developed stigmas of the corresponding female receptors.

Variables evaluated

From a composite sample formed by the physical mixture of the grain from five representative ears per experimental unit, the average length (GL), width (GW) and thickness (GT) of 10 random caryopses were taken, measured with a digital vernier. Similarly, 100 caryopses per experimental unit were counted, which were determined their weight (W100G) in g in an analytical balance and their volume (V100G) in cm³ by displacement of water in a graduated cylinder.

To evaluate the popping characteristics, the harvested grain was brought to an equilibrium moisture between 13.5 and 14% in order to maximize the volume of expansion, which, according to ^{Gökmen (2004}), in popcorn, is achieved at these moisture levels; for this, 40 grams of caryopsis per experimental unit were conditioned in a controlled environment chamber at 21 °C, with a relative humidity of 70% for eight days (^{Ziegler, 2001}).

For the popping, the method standardized by De la O et al. (2015) was used, which consisted of determining the volume of popped corn produced by a sample of 30 g of conditioned caryopsis. The sample was placed in a heat-resistant polypropylene container and processed in a microwave oven (Daewoo^® Kor-164H), set to 70% of its power for 2:45 min, the popped corns that were obtained were placed in a graduated cylinder of 2 L to determine the popping volume in cm³.

The percentage of non-popped caryopsis was calculated with the weight of the non-popped caryopses after heat treatment in the microwave oven over the total weight of the conditioned caryopsis sample (30 g). The type of popped corn was assessed using a visual scale of 1 to 5 according to the way they popped, a value of 1 was used to identify a spherical popped corn (mushroom type) and 5 for an elongated popped corn with bumps (butterfly type). The pulverization of the pericarp, as its disintegration after the popped corn making process, was measured with a visual scale of 1 to 5, where 1 corresponded to a fully pulverized pericarp and 5 to one with little disintegration.

The effect of Xenia caused by the pollen of each of the males on the caryopsis of each of the females for the variables evaluated was estimated using the formula proposed by the authors:

Where: EX= effect of Xenia in percentage on a characteristic of the caryopsis; HM= phenotypic expression of characteristics of the caryopsis of popcorn pollinated with pollen of a male of different type of endosperm; and HF= phenotypic expression of characteristics of the caryopsis of popcorn pollinated with pollen of the same population, equivalent to the female with fraternal cross of male plants of the same type.

Statistical analysis

An analysis of variance was performed using the linear model corresponding to the randomized complete block design. Data on percentage of non-popped caryopsis and visual scales 1-5 were transformed with the formula: arcsine in the analysis of variance. A Tukey mean comparison test at 5% was also applied using the Sas^® statistical package (^{SAS Institute, 2002}).

Variation in physical and popping characteristics of the caryopsis

The analysis of variance detected high significance (p≤ 0.01) between localities (Loc) for three (GL, GW, and pericarp pulverization) of the nine variables (Table 1). This result indicates that the environment has an important effect on the expression of these three characteristics of the caryopsis of popcorn, due to the climatic and edaphic effects typical of the study localities, which includes the sowing dates, agronomic management, the type of soils and the elements of the climate of each locality. These results agree with those reported by ^{Daros et al. (2002}) in a similar work, who observed differences between environments and also detected significant effects of the genotype-environment interaction for caryopsis yield, days to flowering, plant height and popping capacity.

Among females (CA and NAYPP) (Table 1), there were highly significant differences (p≤ 0.01) for caryopsis length, caryopsis thickness, weight of 100 caryopses, percentage of non-popped caryopsis and type of popped corn, indicating that there is high variation for these characteristics among female materials. Among males (M), the effect on female caryopses due to their pollen contribution was highly significant (p≤ 0.01) for GL and popping volume, which suggests that there are differences between males for these two characteristics, so their pollen had a noticeable effect on popcorn (Table 1).

The interaction of females × males (F×M), which can be interpreted as a direct representation of the effects of Xenia, was highly significant (p≤ 0.01) for popping volume, percentage of non-popped caryopsis and pulverization of the pericarp, so it is evident that the type of pollen of the donor and its effect on the caryopsis of the recipient is important in the effect of Xenia, but with markedly differential effect according to the female concerned (Table 1). The interaction of localities × females, (Loc × F) (Table 1) had high significance (p≤ 0.01) for W100G, V100G, popping volume, type of pericarp and pulverization of the pericarp, while for GL and non-popped caryopsis, it was significant (p≤ 0.05), this makes it evident that females behaved differently for these characteristics across the localities, and this could be due to the different genetic response of the materials to the influence of climatic and edaphic factors of the localities, as well as to the agronomic management provided in each of them, such as sowing date, irrigation, fertilization, etc.

On the other hand, significant differences (p≤ 0.05) were only observed for GW and popping volume in the interaction of localities × males (Loc × M) (Table 1), which indicates that the effect of the pollen of each male on GW and popping volume of the caryopses of the females was different across the localities, this as a differential response of the genotype to the contrasting environmental effects of each locality.

F= females; M= males; Loc= localities; Rep= repetitions. The interaction of localities × females × males (Loc × F ×M) (Table 1) was significant (p≤ 0.05) for GW and V100G and highly significant (p≤ 0.01) for popping volume, which is important to highlight, since this variable, in addition to showing its high genetic complexity and complexity of response to the environment, represents both the most valuable and the most desirable characteristic for the production of popcorns.

Table 2 shows the results of the physical characteristics of the caryopsis on average of the localities. It was observed that, in Montecillo, the genetic materials presented statistical superiority in three (GL, GW and W100G) of the five variables evaluated, which again highlights the importance of the effects of the environment on the response of the genotypes, such as climatic and edaphic factors of the localities, as well as those of agronomic management (date of sowing, irrigation, fertilization, etc.). In the comparison of means between localities for the popping characteristics (Table 3), significant statistical differences were observed for type of popped corn and pulverization of the pericarp, a result that agrees with what was reported by ^{Alexander and Creech (1977}) in the sense that the phenomenon of expansion is a polygenetic trait, subject to some influence of the environment.

On the other hand, the comparison between averages of female (F) materials (Table 4) indicated that Compuesto Amarillo produced caryopses of greater length, width, weight, and volume and that NAYPP showed the greatest thickness of caryopsis, this because Compuesto Amarillo contains genetic information of rice-type popcorns, and NAYPP is of the pearl type (rounder grains).

The results also show that the percentage of non-popped caryopsis was not different between CA and NAYPP, but the popping volume was. The type of popped corn was different between the two varieties, as NAYPP produced elongated and slightly protruding popped corns, while CA produced more spherical and medium-sized popped corns.

Table 4 compares the means of each male mated with the two females, which on average measures the influence of each. It can be observed that the physical characteristics of the caryopsis had no significant differences, except for GL, which was diminished by the action of the CML-349 male. On the other hand, significant differences were found in the popping volume, and two different groups formed: 1) the one with the highest popping volume, where the caryopses pollinated with pollen of Compuesto Amarillo and NAYPP, both popcorns, had an increase of 22.08 cm³ and 47.5 cm³, respectively; and 2) the one that was obtained with the effects of pollination with Cacahuacintle and CML-349, non-popcorns of mealy and semicrystalline endosperm, respectively.

Caryopses pollinated with these materials had lower expansion volumes (55 cm³) than when pollinated with CA and NAYPP pollen (84 cm³). These results indicate a clear effect of Xenia, which here was characterized by the decrease in popping volume and that resembles the results obtained by ^{Lyerly (1942}), who observed a reduction of 1.4 volumes in the popping of Yellow Pearl popcorn pollinated with mealy corn, similar results were obtained by ^{Dofing et al. (1991}), observing a decrease in the popping volume when crossing popcorns with toothed corn; for their part, ^{Robbins and Ashman (1984}), when crossing popcorns of inbred lines of the Yellow Pearl race (HP-62-52 and 4722) with inbred lines of crystalline corn (CM80) and toothed corn (B2), found that both generations F₂ and F₃, and crosses of HP62-52 × CM80 (popcorn × crystalline) showed greater volume of expansion than the cross of HP-62-52 × B2 (popcorn × toothed), while generations F₂ and F₃ of crosses 4722 × CM80 (popcorn × crystalline) and cross 4722 × B2 (popcorn × toothed) did not show significant differences in expansion volumes, thus showing that the effects of Xenia have a variable and specific magnitude, depending on the genotypes and environments involved.

The results indicate that the genetic materials, when they acted as males, led to a clear reduction in GL, GW, W100G and V100G in the caryopses of Compuesto Amarillo, as verified when compared with the results obtained when it acted as female. This result is similar to that obtained by ^{Dofing et al. (1991}), where in a similar work, two crosses of popcorn × toothed corn had a decrease in the weight of caryopses. For its part, the NAYPP material was favored by an increase in the aforementioned characteristics, which agrees with the results of ^{Bozinovic et al. (2012}), who observed an increase in the weight of caryopses of a fertile corn hybrid compared to those of its sterile version.

Effects of Xenia for physical and popping characteristics of the caryopsis

When quantifying the effects of Xenia (Table 5), it was observed that for CA the pollen of non-popcorns (Cacahuacintle and CML-349), in general, caused a decrease in the physical characteristics of the caryopsis of Compuesto (GL, GW, GT, W100G and V100G), as well as in the popping volume. This indicates that there was a decrease in these variables due to the effect of Xenia, which did not occur when the female was pollinated with pollen of plants of the same type (fraternal cross). Similarly, the volume of popping and the percentage of non-popped caryopses were disadvantaged when CA was pollinated by males of non-popcorn.

In relation to the type of popped corn, the effect of Xenia was more accentuated with the pollen of Cacahuacintle (-16.66%), as it caused a more spherical type of popped corn in relation to the fraternal cross of Compuesto Amarillo. The effect that NAYPP pollen originated for pericarp pulverization in CA was the most relevant, because it increased this characteristic by 39.28%.

In the case of NAYPP popcorn (Table 5), the results indicate that the effect of Xenia caused an increase in the variables GW, W100G and V100G when this variety was pollinated with the pollen of plants of the same type (fraternal cross) and when the pollen came from Compuesto Amarillo popcorn, there was an increase of 10.5% in the GW variable. In the case of W100G, it was the male Cacahuacintle who caused a more accentuated effect (393.33%) and for V100G, both CML-349 and CAC were the ones that caused a greater increase (18.7%).

The popping volume, as happened in CA, was decreased in equal proportion by both Cacahuacintle and CML-349 pollen (-59%), results that, when they occur in the field, are not favorable for the producer or the consumer. The percentage of non-popped caryopsis increased by 24.48% and 12.49% with pollen of Cacahuacintle and CML-349, respectively, a result that is not favorable because it represents a greater amount of wasted caryopses. The type of popped corn was also modified, going from a unilateral expansion (NAYPP × NAYPP) to a hemispherical one (NAYPP × Cacahuacintle). The pulverization of the pericarp went from being almost intact (NAYPP × NAYPP) to semi-pulverized (NAYPP × CA and NAYPP × CAC).