Introgression breeding through interspecific polyploidisation in lily : a molecular cytogenetic study

Lily has been grown as the most important bulbous crop during the last decade. The genus <em>Lilium</em> consists of seven different sections with about 80 species. Among them, three sections are the most commonly used for breeding cultivars, e.g. Asiatic hybrids of the Sinomartagon section, <em>L. longiflorum</em> of the Leucolirion section and Oriental hybrids of the Archelirion section. The species and cultivars are mainly diploid (2n = 2x = 24) but polyploid forms exist including triploids and tetraploids, which are either induced for breeding purposes or spontaneously found during interspecific breeding programs. Therefore, cultivars include both diploids as well as polyploids of which the latter became more important in recent years.As in all other crops, there is a need for combining desirable characteristics in new cultivars. In lily, such characters are distributed in several species that belong to different sections in the genus <em>Lilium</em> . It is, therefore, essential to hybridise species from different sections and to backcross the F ₁ -hybrids to the cultivars in order to select new cultivars. Generally, it is difficult to hybridise species and to produce backcross progenies, but these crossing barriers can be overcome through integrated pollination and embryo rescue methods. The intersectional species hybrids, however, are highly sterile because of a high degree of genome differentiation of the parents. The main aim of the present research was to develop techniques for using sterile interspecific hybrids in lily breeding more effectively. For this purpose, the F ₁ -hybrids between <em>Lilium longiflorum</em> and <em>L. rubellum</em> ( <strong>LR</strong> ), the amphidiploids of <strong>LR</strong> -hybrid, <em>L. longiflorum</em> and Asiatic hybrids ( <strong>LA</strong> ) and backcross progenies (BC ₁ and BC ₂ ) of these hybrids were analysed through molecular cytogenetic techniques, GISH (genomic <em>in situ</em> hybridisation) and FISH (fluorescence <em>in situ</em> hybridisation).Because it was essential in this study to identify the genomes and chromosomes of the parental species in F ₁ -hybrids and their backcross derivatives, criteria were developed for the identification of individual chromosomes of some of the <em>Lilium</em> species (Chapter 2). For this purpose, the karyotype characteristics based on chromosome length, arm length ratios, C-banding patterns and FISH detection of rDNA sequences (5S and 45S rDNA) was performed. Some of these criteria, together with GISH and FISH were used for the analysis of both mitotic and meiotic chromosomes of the F ₁ -hybrids and their backcross derivatives (Chapter 3, 4 and 5).The diploid (2n = 2x = 24) sterile F ₁ -hybrids were utilised for backcrossing in two different ways, viz., 1) mitotic (somatic) polyploidisation in case of <em>L. longiflorum</em> × <em>L. rubellum</em> hybrids and 2) meiotic (sexual) polyploidisation in case of <em>L. longiflorum</em> × Asiatic hybrids, where 2n-gametes in certain genotypes were formed. A remarkable difference was observed between the two polyploidisation methods. In case of mitotic polyploidisation there was no evidence of any intergenomic recombination between the chromosomes of <em>L. longiflorum</em> and <em>L. rubellum</em> . This was because of the complete preferential pairing between the homologous chromosomes of each species in the amphidiploid (Chapter 3). In the triploid BC ₁ progenies ( <strong>LLR</strong> ) and the aneuploid BC ₂ ( <strong>LLLR</strong> ) progenies there was a total absence of homoeologous recombinant chromosomes (Chapter 3).In case of the 2n-gametes producing F ₁<strong>LA</strong> -hybrids there was clear evidence, through GISH and FISH, for the occurrence of homoeologous chromosome association and recombination as was visible at metaphase I and anaphase I of the microsporogenesis. Most remarkably, these <strong>LA</strong> -hybrids produced three types of 2n-gametes: FDR (first division restitution) without recombination, FDR with recombinant chromosomes and IMR (indeterminate meiotic restitution) with recombinant chromosomes as well as with assortment of homoeologous chromosomes (Chapter 4 and 5).The occurrence of such genetic recombination in these <strong>LA</strong> -hybrids was convincingly proven by analysis of the chromosome composition of the BC ₁ ( <strong>ALA</strong> ) and BC ₂ progenies (Chapter 5 and 6). Thus, it was evident that by using 2n-gametes, as in <strong>LA</strong> -hybrids, sexually polyploidised offsprings can be produced with the addition of complete alien chromosomes as well as with a variable size of recombinant segments. This is advantageous for the introgression of specific characters from alien species into the cultivars. Some triploid BC ₁ progenies ( <strong>ALA</strong> ) derived from crossing with 2n-gametes producing <strong>LA</strong> -hybrids showed a range of fertility (5 - 30 %) and one BC ₁ plant (921238-1) was used as female parent for further crossing, in which many BC ₂ individuals were produced. The strategy of interspecific hybridisation for introgression breeding of lily is now operational by using 2n-gametes producing interspecific hybrids (F ₁ ) combined with backcrossing (Chapter 6).Besides, the practical implications of sexual polyploidisation in lily, the results of the investigations, presented in this thesis, are scientifically significant for many reasons. In the first place, a comparison has been made of the consequences of mitotic and meiotic polyploidisation in this crop. Secondly, in the previous cytogenetic studies the meiotic nuclear restitution mechanisms were rigidly categorised into FDR and SDR. There is now convincing evidence that IMR is a mechanism that combines both FDR and SDR in individual PMC. Thirdly, the molecular cytogenetic study using <em>in situ</em> hybridisation techniques is a powerful tool which enables us not only to create useful breeding material but also to enhance our basic knowledge regarding the modes of origin and composition of polyploids in horticultural crops and species.