Determination of Potential Pollinizer Grafted Pear Hybrids ( Pyrus communis × Pyrus pyrifolia ) for Subtropical Regions in Brazil

The pear tree is originally a temperate-climate fruit tree. The cross Pyrus communis × Pyrus pyrifolia generated hybrid cultivars adapted to subtropical regions. However, the low effective fruiting is a limiting factor for the expansion of pear tree crops in subtropical regions and no pollinizer plants have been identified for these cultivars in subtropical regions. This work aimed to identify possible pollinizer pear tree cultivars in subtropical regions by evaluating the stigmatic receptivity, the germination percentage, and the number of pollen grains. Seven hybrid pear cultivars were used. Flowers of each cultivar at each floral stage were collected (E, E2, F, F2, and G). The stigmatic receptivity was evaluated by immersing the material in 3% hydrogen peroxide. Flowers were collected for the quantification of anthers, pollen grains per anther and per flower, and pollen grain germination. Stigmas were 100% receptive for all cultivars at the F2 and G stages. Cultivar Cascatense showed a large number of anthers and pollen grains per anther and per flower. The highest pollen grain germination percentage was detected for Cascatense (72.75%). Cultivars Seleta and Triunfo have high stigmatic receptivity for a longer period during the flowering stage, proven to be a potential female parent. Cascatense has a higher germination percentage and a larger number of anthers per flower, pollen grains per anther, and pollen grains per flower. Cascatense has a high potential to be used as a pollinizer, both in pear tree breeding programs and commercial orchards.


Introduction
Despite being a temperate-climate fruit tree, some pear tree hybrid cultivars obtained from the cross Pyrus communis × P. pyrifolia are adapted to subtropical regions, where the climate is characterized by mild winter and higher temperatures in the summer, compared to temperate regions (Curi et al., 2017).The cultivation of these hybrid cultivars in subtropical regions was made possible by the combination of the quality of the European pear (P.communis) with the low chill hours required by the Asian pear (P.pyrifolia) (Chagas et al., 2008).
Most pear tree cultivars have gametophytic self-incompatibility, causing the plant to reject its own pollen.Therefore, they depend on cross-pollination for fruit production (Franklin-Tong & Franklin 2003).In general, the use of two to three pear cultivars with a coincident flowering period is recommended for commercial crops.The low effective fruiting due to lack of pollinizer plants, is a limiting factor for the expansion of pear tree crops in subtropical regions (Bettiol Neto et al., 2014).
The absence of the ovule fertilization in pollinated flowers, in both temperate and subtropical regions, results in a small number of fruits set per tree, resulting in reduced fruit yield.Fewer seeds were formed under insufficient pollination condition due to limited supply of phytohormones for ovary and seed development (Tavares et al., 2002).
To overcome the gametophytic incompatibility and provide adequate fertilization for increasing the effective fruiting in pear orchards, pollen grains of genotypes compatible with favorable "S" allele series must be transferred by pollinator insects (Sezerino & Orth, 2015).Due to the self-incompatibility in pear cultivars, pollinizer plants are used; however, they must be compatible and provide a large number of pollen grains to the main fruiting cultivar (Nakasu & Faoro, 2003).The flowering time and flowering stages of the pollinizer cultivar and the recipient cultivar must be synchronized.Moreover, the pollinizer tree must produce viable pollen in adequate quantity and quality to guarantee fruit production (Bettiol Neto et al., 2014).
The receptive stigmatic surface and the duration of its availability is fundamental to determine the best period of pollen deposition on the flower.For successful pear fertilization, the pollen grain must present high viability and germination rate (Brito et al., 2010).
The in vitro pollen germination test is essential for breeding programs as they allow verifying pollen viability (Ramos et al., 2008).Pollen grain viability and germination are crucial factors to determine fruit set, and they influence fruit yield (Irenaeus & Mitra, 2014).
There is a wide variation among the cultivars within the same species in terms of pollen viability, germination and the specific requirements for the reproductive process to occur (Irenaeus & Mitra, 2014).
Therefore, this study aimed to identify possible pear tree pollinizer pear cultivars in subtropical regions by evaluating the stigmatic receptivity, the pollen germination percentage, and the number of pollen grains.

Material and Methods
The plant material was collected during 2015 and 2016 in Lavras, Brazil located at latitude 21º14′ S, longitude 45º00′ W, and at an altitude of 841 m above mean sea level.The climate of the experimental site is classified as Cwa (subtropical climate, with cold and dry winter and hot and humid summer) (Souza et al., 2017).Seven Pyrus communis × Pyrus pyrifolia hybrid cultivars were evaluated: Cascatense, Centenária, D'água, Primorosa, Seleta, Tenra, and Triunfo (Table 1).The hybrid cultivars were grafted on to the chosen rooststock Pyrus calleryana and the graftlings were transplanted to the field in October 2010.Plants were spaced at 3.0 × 4.0 meters (833 trees/ha) and conducted in a modified central leader system.The management of trees followed modified central leader system for pruning, standard fertilization, phytosanitary control as recommended for pear tree.

Stigmatic Receptivity
Stigmatic receptivity was verified by the viscous and wetting aspect of the stigma (Almeida, 1986) and tested with 3% hydrogen peroxide (H 2 O 2 ) (Kearns & Inouye, 1993).Flowers were collected at different floral development stages.Forty flowers of each cultivar at each floral stage were collected (E, E2, F, F2, and G-Figure 1).The material was randomly divided into four replications and ten flowers per replication.
Drops of 3% hydrogen peroxide were applied to the stigmas of the collected flowers.Those that showed bubble formation were considered as receptive.The percentage of receptive stigmas was evaluated at each floral stage by counting the number of stigmas that had bubble formation.At each floral stage, the number of receptive stigmas was multiplied by 100 and then divided by the total number of stigmas evaluated in this case, ten stigmas to calculate the percent receptivity of stigma.Data were subjected to analysis of variance, and the groups of means were compared by the Scott-Knott test at 5% probability. Figure

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For successful pollination, the pollen transferred to the receptive stigma must germinate, leading to fertilization.In many cases, fertilization may also occur when the pollen grain is deposited before the stigmatic receptivity period, provided that it remains viable to germinate as soon as the flower becomes receptive (Ramos et al., 2002).Under stress conditions, the stigmatic receptivity period may be affected due to a decrease in the amount of exudates (Srinivasan et al., 1999).

Number of Anthers per Flower and Pollen Grains per Anther and per Flower
Statistical analyses revealed a significant difference between cultivars regarding the number of anthers per flower, pollen grains per anther, and pollen grains per flower (Table 2).Accuracy estimates indicate the precision with which the experiments were conducted and the existence of variability.Appropriate accuracy values are close to the unit or 100% (Resende & Duarte, 2007).In the present study, for all evaluated traits, the accuracy was higher than 95%, confirming the accuracy of the evaluated data.Note.*Means followed by the same letter in the column belong to the same group by the Scott-Knott's test at 5% probability.The abbreviated term "No." indicates "number".
Cascatense has the largest number of anthers per flower, pollen grains per anther, and pollen grains per flower, simultaneously, showing potential to be used as a pollinizer.Triunfo has a large number of anthers per flower but a very small number of pollen grains per anther, resulting in a low quantity of pollen grains per flower.Pear trees require cross-pollination due to the gametophytic incompatibility.The production of large amounts of pollen grains is a desirable trait since it increases the probability of cross-pollination (Sezerino & Orth, 2015).
Cultivars Centenaria, D'água, Primorosa, Tenra, and Seleta showed less than 20 anthers per flower (Table 2).This result may be related to a trait inherited from one of their parents.Nogueira et al. (2015), working with cultivar Packham's Triumph, detected 12 anthers per flower.Cultivars Seleta and Tenra showed a small number of pollen grains per anther and per flower.This result may be related to the lower adaptation of these cultivars to high-temperature regions.Nava et al. (2009) studied peach trees and verified a decrease in the production of pollen grains per anther due to high temperatures and water stress, which may have influenced the floral organogenesis and microsporogenesis stages.
The difference in the number of pollen grains per flower might be related to the fact that temperate-climate fruit trees require high chill hours.Therefore, when these cultivars are cultivated in regions with low chill hours, they can produce smaller flowers, smaller anthers, and fewer pollen grains than those cultivated under extended periods of cold or low temperatures during pollen development in temperate climatic zones compared to subtropical regions (Nogueira et al., 2016).

Percentage of Pollen Grains Germination
Statistical analysis of the results revealed a significant difference between the pear tree cultivars in relation to the percentage of successfully germinated pollen grains (Figure 3).The clustering of cultivars based on mean data on this per cent germination trait, resulted in four distinct clusters.The highest percentages of pollen grains germinated were observed in Cascatense (72.75%), followed by Primorosa (59.90%) (Figure 3).The percentage of pollen grains germinatedin Tenra and Triunfo was not detected and the data was not presented in the figure 3 and Figure 2 s anthesis.F when the f Figure Note.* m probability

Table 1 .
List of cultivars with their origin and genealogy

Table 2 .
Mean number of anthers per flower, mean number of pollen grains per anther and per flower in different grafted pear tree hybrid cultivars CultivarNo. of anthers per flower No. of pollen grains per anther No. of pollen grains per flower