Tools for hybrid breeding in industrial chicory (Cichorium intybus L.)

Ondertitel:Technieken voor de hybride veredeling van industriële cichorei (Cichorium intybus L.)

Industrial chicory (Cichorium intybus L.) roots are mainly grown for the extraction of the prebiotic soluble dietary fiber inulin. A small percentage is still used when dried and roasted as a coffee surrogate or additive. Most current cultivars are synthetic cultivars based on four to ten mother clones. To enhance yield and uniformity, F1 hybrid breeding is desired. For this there are two main prerequisites: homozygous lines and a controlled pollination system. First, homozygous lines can be acquired by inbreeding through self-pollination. As industrial chicory is a biannual crop, this is a time consuming process. Also, as a result of pseudo self-incompatibility, line development can be challenging. To circumvent the time consuming inbreeding process, doubled haploids can be used. In a first step, haploid plants containing only one basal chromosome set (2n = x = 9) instead of two copies of each chromosome as in diploids (2n = 2x = 18), are regenerated. Upon chromosome doubling of these haploid plants, 100% homozygous doubled haploids are obtained. This procedure takes the time of a single sexual generation and thus dramatically reduces the time required for homozygous line development. Second, a controlled pollination system is necessary to prevent self-pollination during seed production and ensure only hybrid seeds result from the cross of two different homozygous lines. There are different systems to prevent self-pollination, but cytoplasmic male sterility (CMS) is preferred by breeders. This type of male sterility is maternally inherited and can be maintained by pollination with isonuclear lines. It is frequently caused by transcripts of a novel mitochondrial open reading frame. In this thesis, we assessed different haploid inducing protocols. Anther culture and microspore culture were performed in an attempt to initiate embryogenesis in male gametophytes. Likewise, ovule cultures were started to provoke regeneration from female gametophytes. Additionally, intergeneric hybridization with related species was accomplished. Only crosses with Cicerbita alpina resulted in haploid plants. In total, 18 haploids regenerated after 2826 crosses. Two chromosome doubling protocols, one using mitotic inhibitors and another based on protoplast regeneration were successfully developed. Beside haploid plants, true hybrids between chicory and Cicerbita were obtained. We hypothesized a system of chromosome elimination after intergeneric crosses with Cicerbita alpina. In literature, a similar mechanism was demonstrated in the cross between Hordeum vulgare and Hordeum bulbosum from which barley haploids are induced. In this model, proteins loading on the centromeres play an essential role. The most important, centromeric histon H3 (CENH3) is loaded less efficiently on the paternal chromosomes. Therefore we speculate that Cicerbita alpina chromosome elimination may act, at least in part, through a mechanism based on asymmetric CENH3 functionality and/or sub-genome specific centromere binding. We tested temperature stability of alloplasmic CMS chicory genotypes generated by pollinating a wild chicory type with industrial chicory and subsequent multiple backcrossing with industrial chicory. Visual scoring of different CMS clones in different temperature conditions indicated a temperature sensitivity. The early floral bud stage was sensitive to high temperatures, resulting in fertile flowers 12 – 17 days after exposure. Experiments in controlled growth chambers simulating a heat shock confirmed that short periods of high temperature during early floral buds development could induce pollen production We examined the development of wild-type floral buds by a microscopical study and trancriptome analysis. The latter yielded several sets of differentially expressed genes during floral bud growth. The combined data from microscopical cross sections and the number of differentially expressed genes allowed us to propose the critical phases in bud development. Subsequently, using again morphological and transcriptomic data, WT development was compared to CMS development. We considered as well the number of genes that had an altered expression profile in CMS lines as the expression of particular genes whose orthologues were relevant for male fertility in other crops according to literature. Aberrant tapetum development was observed in CMS anthers. In some cases, gene expression was upregulated either sooner or later in CMS lines than in WT plants; also, for particular genes such as CiAMS, CiINV4 and CiEXI1 the maximal expression clearly differed in WT and CMS plants. As a proof of concept, a field trial of hybrids obtained by a cross between a partially inbred CMS seed parent and a DH pollen parent showed the potential of hybrid breeding. The inulin yield had increased by 15% compared to a reference synthetic cultivar.

ISBN:9789463572279

Jaar van publicatie:2019

Toegankelijkheid:Open