Pre-harvest Sprouting Tolerance of Triticale Genotypes in Brazil

Pre-harvest sprouting (PHS) represents one of the main factors, which causes yield, technological and physiological losses in triticale seeds (X Triticosecale Wittmack). This work aimed to rate the variability and identify potential pre-harvest sprouting tolerant sources in triticale genotypes. Based on that, 32 triticale and three wheat genotypes were sown in 2016, 2017 and 2018 growing seasons, in Londrina-PR, Brazil. After the ears harvesting, these were submitted to simulate raining, for sprouting induction, through nebulization in a greenhouse. After nebulization, ears were sun dried, later hand threshed to determain, grain germination percentage (GERM) and hectoliter weight (HW). Additionally, it was determined grains HW of ears, whom were not submitted to nebulization, as well as the whole meal flour falling number (FN). The experiment design was completely randomized design, with two replications, and the experimental unit was made of 20 ears. The data collected were subjected to analysis of variance (ANOVA) and Scott-Knott test. Frontana, ND 674 and Quartzo are a source of tolerance to PHS in wheat. In triticale genotypes, genetic variability was observed for GERM, FN, and HW prior and after nebulization. The triticale genotypes BRS Netuno, BRS Saturno, TCL 15116, X 092181, Tiguera 1 and Tiguera 8, where tolerant towards PHS.


Introduction
The triticale (X Triticosecale Wittmack), originated from the hybridization of wheat (Triticum) with rye (Secale), aiming to unite favorable traits of both these species. However, triticale cultivars still have deficiencies in some agronomic traits, like susceptibility to certain diseases (fusarium head blight, blast, leaf spots and viruses) (Mergoum et al., 2019).
In 2018, the harvest areas and production of triticale wordwide were 3, 809,192 hectares and 12,802,592 tons, respectively (FAO, 2020). Poland, Germany, France, Belarus, China, and Spain are the leading triticale producing countries (FAO, 2020). Triticale can be used for several purposes. Triticale is used in animal feed, it is used especially for direct feeding as green forage or as silage of young or mature plants, moist or dry grains, and hay (Nascimento Junior et al., 2004). In human feeding the triticale grains, its flour has been used to compose mixtures with wheat flour for the manufacture of low fermentation products, such as cookies, pizza dough, meatballs, waffles, and cakes (Watanabe, 2016). Triticale has also aroused interest for biofuel production, and it has presented competitiveness when compared with other winter cereals (McGoverin et al., 2011).
Pre-harvest sprouting (PHS), is a premature grain sprouting in the ear, before the harvested. This problem has been observed over many years in several triticale and wheat growing areas, all over the world. This represents a limiting technological quality factor, to the processing industry and seed production physiological quality (Moś & Wójtowicz, 2004;Chapman, 2011). In general, dormant grain happens under cool growing conditions, whereas high temperatures during the later stages of grain growth, break embryo dormancy allowing grains to germinate in the ear, granted rain occurs around harvest time.
The ears were taken to a greenhouse for nebulization. This method allowed an artificial rain simulation, similar to what occurs in the field. Ears were exposed to nebulization for 42 hours, 68 hours and 42 hours in 2016, 2017 and 2018 growing seasons, respectively. This method has been utilized successfully in the main wheat breeding programs in Brazil, for pre-harvest sprouting resistant genotypes selection (Franco et al., 2009;Okuyama et al., 2017). The experiment design was completely randomized, with two replications, being the experimental unit made of 20 ears.
Grain percentage sprouting (GERM), was determined from ears submitted to nebulization. After nebulization, ears were sun dried and later, hand threshed. GERM was determined based on assessment of two random 50 grain samples on each plot. Germinated grain are those with visible vegetative structure or even visible pericarp breaking.
The FN (also known as Hagberg Number) was determined in samples (7 g) of ground wheat, which were mixed with 25 mL of distilled water in a FN tube with a stirrer and shaken to form slurry. This slurry was heated in a boiling water bath at 100 °C and stirred constantly. The time it took the stirrer to drop through the paste was recorded as the FN value in a Perten FN 1500 (Perten Instruments), according to the 56-81 B method of the American Association of Cereal Chemists (American Association of Cereal Chemists [AACC], 2000).
Hectoliter weight was determined from two sources, from grains which were not submitted to nebulization (HW_s) and from grains originated from ears following nebulization (HW_n). verified, joint variance analysis was perfomed, considering genotypes and years effects as fixed. Scott Knott test at 5% probability was carried out for all traits.
Pearson correlation coefficients were estimated to observe the magnitude and direction between trait pairs. Correlations significance was done through t test with two freedom degrees. Statistical analyses were done with the Genes software aid (Cruz, 2016). Table 1 demonstrates the variance analyses results for every assessed trait. Significant effect for genotype (p < 0.05) was observed for all assessed traits. This result indicates the existence if the differences between the evaluated genotypes for hectoliter weight prior and following nebulization, falling number and ear with germinated grains. Considering unfolding within the genotype effect, triticale's significant effect (p < 0.05) was observed, indicating performance differences among the genotypes, which allow a selection of superior genotypes. The control effect was not significant (p > 0.05), for ear sprouting percentage (GERM), an anticipated fact, as Frontana, Quartzo and ND 674 wheat genotypes, utilized as controls, are rated as tolerants to pre-harvest sprouting. The group significant effect (p < 0.05) reveals that there is a difference among triticale vs wheat groups towards the assessed traits. Note. HW_s = hectoliter weight prior nebulization (kg hl -1 ); HW_n = hectoliter weight following nebulization (kg hl -1 ); FN = falling number (seconds); GERM = sprouting seed (%). ns not significant by the F test; ** Significant (p < 0.01) by the F test; * Significant (p < 0.05) by the F test.

Results and Discussion
A significant effect between growing seasons (p < 0.01) for all traits, was detected, indicating that the growing conditions may influence genotypes response. All trials were conducted in the same location, but in different agricultural years. Environmental conditions varied from year to year, this fact can be seen in Figure 1. A significant effect (p < 0.01) for the double interactions Genotype × Year, Control × Year and Group × Year was also detected, suggesting differentiated genotype behavior in the different growing seasons (Table 1). Only the interaction Control × Year for the trait GERM was not significant, since all controls utilized in this study present pre-harvest sprouting tolerance and presented behavior stability on the three years assessed.
Environmental variation coefficients estimates (CV) varied on 0.3% (hectoliter weight prior nebulization) to 24.8% (pre-harvest sprouting percentage), which represents good experimental precision for this study according to Gomes (2000).
The occurrence of harvest raining affects HW negatively, since all genotypes presented HW reduction when submitted to nebulization (Table 2). Due the simulated raining, an average reduction of 9.4% in the HW (Table 2) was verified. A HW magnitude of reduction due to simulated raining, was distinct among triticale and wheat genotypes (Table 2). Triticale cultivars BRS Saturno, BRS 203 and lines TCL 15052 and TCL 15022 presented the lowest HW reductions, following nebulization. On the other side, genotypes TCL 15102, TCL 11014, ITW 11014 and IPR Aimoré were the most effected, since they presented the greatest HW reductions.
Wheat genotypes are set at the greatest tolerance class towards sprouting in all the assessed years (Table 3).
Wheat cultivar Frontana, known as a pre-harvest sprouting tolerant source, is much utilized as a parent on breeding programs in Brazil. On the study done by Franco et al. (2009), they reported that dormancy seems to be the most evident mechanism, since the greatest number of dormant seeds detected trough tetrazolium test were associated to the lowest sprouting rates.
Pre-harvest sprouting tolerant cultivars breeding is one of the main challenges to the triticale breeding in the country. The comparative study among the triticales highlighted six promising genotypes: BRS Netuno and BRS Saturno cultivars, TCL 15116 and X 092181 lines and the accesses Tiguera 1 and Tiguera 8. These genotypes had been inserted at the same pre-harvest sprouting control tolerant groups (Frontana, ND 674 e Quartzo) and have presented behavior stability, showing low sprouted grains value on the three years assessed (Table 3). Tolerant genotypes identification permit its use as parents in breeding programs aiming to get future cultivars with great pre-harvest sprouting tolerance levels.
The presence of the Genotype × Environment (G × E) interaction makes it difficult to classify genotypes with intermediate HWS resistance. The triticale lines TCL 15022, TCL 15020 and TCLD 1003 were classified as moderately resistant in one season and as moderately susceptible in another season (Table 3).
ITW 11014 and TCLD 0903 were pre-harvest sprouting susceptible, due to the high sprouting values found in the three years of assessment (Table 3). Pre-harvest sprouting is a serious grain quality concern for millers and farmers alike. Sprouting happends as the seed breaks dormancy, and activates enzymes that begin breaking down starch.
Falling numbers is a test that indicates how well the grain's starch has retained its strength. The FN of the triticale genotypes were lower than the wheat genotypes (Table 3). The FN in the triticale genotypes ranged from 62 seconds to FN > 250 seconds. Climatic conditions influenced this characteristic in different growing season. In 2017, the FN average of triticales was 188 seconds. The average FN in 2016 and 2018 was 99 and 86 seconds, respectively. The higher average FN in 2017 is probably due to absence of rainfall at the end of the grain filling and maturation period ( Figure 1).
Falling number trait does not demonstrate correlation with pre-harvest sprouting grains percentage (GERM) on triticale genotypes (Table 4). Such an issue might be explained, by the fact that, triticale genotypes scored the minimum FN the equipment "Falling Number" can register. Triticales' FN cannot be directly compared to wheat, because factors besides α-amylase decrease triticale' solution viscosity a lot. What would be considered a low wheat FN number, not necessarily indicates high α-amylase activity in triticale. A possible cause is the endogen enzymes action, which does not have α-amylase, particularly those which act on non-starch polysaccharides (Randhawa et al., 2015).
A high correlation was detected, although negative, between pre-harvest sprouting grains percentage (GERM) and nebulized grains hectoliter weight (HW_n) on the three assessed years (Table 4). Therefore, grain HW quantifying following simulated rain may be used to the aid screening pre-harvest sprouting tolerant triticale genotypes.