Reproductief success en polyploïdisatie capaciteit in banaan (Musa spp.):Invloeden van genetische, ontwikkelings- en omgevingsfactoren.

Banana (Musa spp.) is the worlds most produced and consumed fruit crop, grown in tropical regions for fresh fruit consumption or further processing into storable foods. Despite its large importance to both international trade and smallholder farmers in developing countries, banana cultivars have not seen any major genetic improvements since their domestication. This is due to a particular trait of edible bananas, their lack of seed formation. Cultivated bananas have resulted from several hybridizations between Musa species and subspecies, resulting in significant structural heterozygosity, associated with reduced fertility and spontaneous diploid (2n) gamete formation, resulting in the formation of triploid plants that were selected for their improved fruit quality over diploid accessions, and their almost complete sterility. Though seedlessness is necessary to allow consumption of banana fruits, it has major consequences. As these seedless plants are continuously vegetatively propagated, there is a lack of genetic variability within cultivars, which renders them extremely vulnerable to changing climates and the increasing drought in tropical regions in particular, as well as the emergence of new diseases. Improved banana varieties with enhanced resilience to (a)biotic stresses are therefore highly needed. However, sexual recombination between parental lines and selection of progeny form the foundation of genetic improvement through breeding, which is nearly nonexistent in triploid bananas. Nevertheless, current banana breeding programs use the little residual female fertility in existing triploid cultivars to cross these with improved diploids carrying resistance traits. Occasionally this yields tetraploid progeny, which is again crossed with an improved diploid to yield improved triploid cultivars. With millions of pollinations being performed to obtain a handful of progeny, this breeding approach is highly inefficient. Low seed set in banana is a complex issue, with numerous factors contributing to the overall reproductive failure. These include suboptimal pollen and ovule viability, often linked to genomic imbalances between parental lines, limited receptivity of the female stigma, improper timing of pollination, insufficient pollen tube growth and fertilization, as well as developmental arrest of embryos following fertilization. While all of these factors are thought to decrease reproductive success, the underlying mechanisms and relative contribution of these defects to the seed formation bottleneck require further investigation in order to design strategies to increase the success of crosses. Next to the classical breeding approach, an alternative breeding approach is explored, via improvement of ancestors of current triploid landraces at the diploid level, with the induction of triploid sterility through unilateral sexual polyploidization at the very end of the breeding process. This approach benefits from the higher fertility of diploid accessions, and the more straightforward inheritance of traits. One established method to achieve this involves chemically induced genome doubling of diploid parents, generating tetraploid plants capable of producing diploid (2n) gametes. However, these 2n gametes tend to possess little recombination due to the genome doubling, as a consequence, the resulting triploid offspring typically shows low genetic diversity, which limits the genetic gain that can be obtained. As an alternative, induction of 2n gamete formation via external treatments that trigger meiotic restitution offers a promising and underexplored strategy for ploidy breeding in banana. In this technique, 2n gametes can be obtained directly from selected diploid parents, omitting the tetraploid generation needed in both of the aforementioned breeding approaches, thereby saving substantial time. Additionally it allows for optimal use of the genetic resources through control over the hetero- or homozygosity of the resulting 2n gametes. In the first part of this PhD research, we explored various hybridization barriers in banana with the aim of identifying opportunities to enhance crossing efficiency. Our investigation began with an analysis of male meiotic stability across diploid, triploid, and tetraploid Musa genotypes, focusing particularly on the natural occurrence of 2n pollen, an important factor in banana domestication. We found that meiosis was relatively stable in wild diploids, whereas diploid cultivars showed more anomalies, mostly involving the formation of aneuploid microspores. Out of the analyzed diploid cultivars, only 'Pisang Lilin' showed considerable amounts of meiotic restitution and higher ploidy pollen grains. Triploid and tetraploid varieties consistently showed meiotic defects resulting in aneuploid spore formation, the severity and nature of meiotic errors varied between genotypes. We then examined the viability and size, as indication for ploidy, of mature pollen grains during male flowering in multiple diploid and triploid genotypes. This analysis demonstrated that both the developmental stage of the inflorescence and environmental conditions during microsporogenesis significantly influence pollen viability. In wild Musa accessions, pollen viability and germination capacity gradually decline over the course of inflorescence development. Additionally, high radiation exposure prior to pollen mitosis I and II significantly reduced germination capacity, while elevated radiation during meiosis negatively impacted pollen viability. In contrast, the Musa cultivars examined in this study exhibited a positive correlation between the developmental age of the male inflorescence and pollen viability, germination capacity, and pollen diameter. Elevated temperatures during meiosis were found to enhance pollen size, viability, and germination percentage. Additionally, we investigated pollen tube growth dynamics through a series of controlled crosses involving both wild diploid Musa accessions and cultivated diploid and triploid varieties. The effects of pre-anthesis pollination and the timing of pollination during the day (morning vs. evening) on pollen tube progression were also examined. Our findings highlight pollen retention and limited pollen tube progression as significant pre-zygotic hybridization barriers in Musa cultivars. Nonetheless, the occasional success of pollen tubes reaching the ovary indicates potential for improvement. Pollination prior to anthesis consistently resulted in lower pollen adhesion and fewer, slower-growing pollen tubes. Similarly, evening pollination using immature pollen led to diminished pollen tube growth. However, when mature pollen was applied to pistils at anthesis, no significant differences were observed between morning and evening pollination. Based on these observations, we recommend to restrict breeding crosses to post-anthesis flowers to enhance pollination efficiency. Further investigation of ovule development in both open- and hand-pollinated diploid and triploid plants revealed a pronounced barrier to seed formation, with no embryo or seed development observed in any of the tested cultivars. In the triploid cultivar, all ovules seemed to be non-functional, indicating the primary seed-formation barrier in this case to occur before putative fertilization. In the diploid cultivar, 35% of ovules appeared non-functional, while the remaining 65% exhibited enlargement for up to two weeks after anthesis before eventually shriveling, similar to the behavior of unfertilized ovules in wild diploids, suggesting a failure of fertilization to be the main seed-formation barrier in this genotype. In the second part of the thesis, we evaluated the potential of different techniques to induce 2n gamete formation in Musa species. Our findings show that meiotic restitution can be effectively triggered by temperature stress (both cold and heat) and by gibberellic acid treatments. Following heat treatment at 38°C, the formation of anaphase bridges caused by nonhomologous recombination, inhibited cytokinesis during both meiosis I and II. Similar, after exposure to 4°C and gibberellic acid treatments, the presence of lagging chromosomes during meiosis I and II likely also disrupted cytokinesis. As a result, both FDR- and SDR type 2n gametes are expected. Temporal mapping of meiosis in banana and further finetuning of the induction techniques should allow to steer towards the preferred restitution type. These methods represent promising tools for inducing 2n gametes in banana, offering a potential route for bypassing some of the reproductive limitations in current breeding strategies. In conclusion, this PhD thesis has provided new insights into the complex network of reproductive barriers that contribute to sterility in banana. By characterizing these barriers and demonstrating the feasibility of inducing 2n gametes, this work lays the foundation for alternative approaches to triploid banana breeding. Our findings contribute to a better understanding of fertility dynamics in Musa spp. and open new possibilities for exploiting residual fertility in edible bananas for genetic improvement.