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Self-incompatibility and the use of gibberellins in pear fruit production (Pyrus communis L.)

Book - Dissertation

Unpredictable fruit set and poor yield are important problems in commercial pear production worldwide and are often the result of a combination of self-incompatibility and poor (insect) pollination. The S-RNase dependent self-incompatibility mechanism in pear inhibits growth of genetically incompatible pollen tubes and thereby prevents fertilization and fruit set after self-pollination. In order to ensure adequate fruit set, growers may aim to facilitate cross-pollination in their orchard by planting compatible pollen donor cultivars within the rows of the main cultivar and/or by promoting the presence of pollinating insects either through the placement of honey bee or bumble bee hives or through the improvement of nesting quality for wild (bee) pollinators. However, even after such efforts, pollination and fertilization of pear flowers may still be hampered by suboptimal weather conditions during early pear bloom. Therefore, many growers use plant growth regulators (PGR) such as gibberellic acid (GA) to stimulate parthenocarpic fruit set which is independent of fertilization. Gibberellic acid was first introduced as an effective curative treatment to stimulate fruit set after a frost event during flowering. Today, it has become a standard procedure, especially in Belgium, although the effectiveness of GA treatments is still debated in non-parthenocarpic cultivars and in the absence of frost. The aim of this thesis is to expand the current knowledge of the female S-determinant of the Pyrus self-incompatibility system, namely the S-RNase, as well as to deliver meaningful insight into how gibberellin treatments affect fruit set, development and quality with a specific focus on the two most relevant Belgian cultivars, namely the parthenocarpic cultivar "Conference" and the non-parthenocarpic cultivar "Doyenné du Comice". The first part of this thesis focuses on the self-incompatibility mechanism in pear. A new assay based on the semi in vivo method in Arabidopsis thaliana was developed to quickly and accurately determine the compatibility of a cross between two cultivars. In this method, pear flowers are hand pollinated and incubated for 3h in vivo, after which the styles are cut at half-length and incubated in vitro for 48h. At this time, the number of pollen tubes that has grown through the style and into the medium is counted and compared to the number of emerging pollen tubes after a reference compatible and incompatible cross. Since the number of pollen tubes differs significantly between cross-compatible, semi-compatible and self-pollination treatments, it is a suitable parameter to accurately determine the specific compatibility type between two pear varieties. The added value of this method over existing techniques is the high reproducibility and the increased accuracy, specifically with respect to distinguishing cross- and semi-compatibility. Next, we determined the full sequence of the S-RNase alleles of 50 Pyrus communis cultivars, 3 Pyrus pyrifolia cultivars, 2 wild Pyrus accessions and 1 hybrid cultivar and additionally SNP-fingerprinted all included diploid cultivars. The sequencing results revealed two previously undescribed S-alleles. Using the previously developed semi in vivo method, one new S-allele was shown to probably be functionally different from the most similar, known S-allele (99.1% identical sites based on the predicted amino acid sequences). Additionally, several synonymous mutations leading to neutral variants of the same S-allele were discovered, with important implications for our current understanding of the evolution of new S-allele specificities. In the second part of this thesis, the effects of gibberellin treatments in "Conference" and "Doyenné du Comice" cultivars were assessed regarding fruit set, fruit growth, yield and fruit quality. Our results show that gibberellins can increase fruit set and yield in "Conference" if sufficient thinning is performed. However, the same increase in fruit set was found after self-pollination, suggesting that more attention for pollinating insect pollinations in the orchard may improve fruit set and yield in the same amount as yearly gibberellin treatments without the associated negative consequences regarding fruit shape, fruit size and return bloom. Cross-pollination was the best way to increase yield. For "Doyenné du Comice" the effects of gibberellin treatments were very limited and cross-pollination was the only treatment that led to consistently high yields. The occurrence of severely elongated pears, also known as "bottle shaped pears" in "Conference", which is often attributed to gibberellin applications, was also investigated. Our results revealed that bottle shaped fruit is a natural occurrence in "Conference" and several other cultivars. They result from the apical flower in the flower cluster and are therefore not directly caused by gibberellin application, although gibberellin application increases the frequency of these natural deformations by inhibiting the natural abscission of the apical flower in these cultivars. Finally, both main research topics of this thesis were combined in a final experiment where we measured the effect of gibberellin treatment, as well as the different types of pollination (self/semi-compatible/cross-compatible and no pollination) on the allele-specific expression of the S-RNase gene in "Conference". Our results revealed that pollination influences the temporal expression pattern of both S-RNase-alleles in the "Conference" pistil suggesting the presence of a yet undescribed regulatory mechanism in the self-incompatibility system of Pyrus. In contrast, we could not find evidence of any noteworthy effects of a single GA4+7 treatment (10 ppm) on S-RNase expression. In conclusion, in this thesis, the full coding sequence of the S-RNase alleles of 56 pear cultivars was determined, revealing the presence of intra-allelic variation and the existence of two previously unknown S-RNase allele specificities. One new S-RNase allele was functionally assessed using a semi in vivo pollination assay that was optimized for pear in this thesis. We also found that the type of pollination can affect transcript levels of the S-RNase in the style, although more extensive follow-up experiments are necessary to better understand the underlying molecular mechanisms. Our results provide a strong basis for follow up research focused on the self-incompatibility mechanism in pear and for possible practical applications such as the development of self-compatible cultivars. In the absence of these self-compatible cultivars, gibberellin treatments remain the standard method to artificially improve fruit set in commercial pear production. However, results presented in this thesis further question the usefulness of these applications in the absence of frost, especially in non-parthenocarpic cultivars like "Doyenné du Comice". In addition, we were able to demonstrate that gibberellin treatments are not the cause of elongated "bottle shaped" fruit in "Conference" although they do increase their occurrence. This insight is an important step in the better understanding of the negative impacts of gibberellin treatments and can contribute to more efficient thinning methods as well as the development of improved gibberellin formulations or application procedures, although further research on this topic is necessary.
Publication year:2021
Accessibility:Open