MEIOTIC POLYPLOIDIZATION IN FIVE DIFFERENT INTERSPECIFIC LILIUM HYBRIDS

With the aim of transferring important horticultural and disease resistance characters that are available in different Lilium species (2n=2x=24) into the cultivars of lilies, an extensive interspecific hybridization program is in progress at the Plant Research International, Wageningen. In this context, we have made thousands of interspecific hybrids, and investigated them for the occurrence of 2n gametes. These sexual polyploid progenies that were produced were cytologically analyzed through genomic in situ hybridization (GISH). Out of the five different sets of interspecific hybrids that produce 2n-gametes, the following four hybrids involved species that belonged to different taxonomic sections: L. longiflorum x Asiatic hybrids; L. longiflorum x L. henryi; Oriental x Asiatic hybrids and L. auratum x L. henryi. One was an intra-sectional species hybrid involving the cultivar ‘Enchantment’ x L. pumilum. In all the four intersectional hybrids meiosis was highly irregular during meiosis and produced almost exclusively first division restitution (FDR) gametes. On the contrary, the single hybrid, ‘Enchantment’ x L. pumilum had normal chromosome pairing and produced only second division restitution (SDR) gametes. Although the frequency of 2n-gamete production in all cases was greatly influenced by the environment, it was possible to produce backcross progenies in all cases. Analyses of backcross progenies through GISH proved that intergenomic recombination occurred in the case of FDR gametes that were produced in all the four interspecific hybrids. Furthermore, analyses of BC2 and BC3 progenies in some cases proved that recombinant chromosomes were transmitted normally. These investigations have shown that meiotic polyploidization is highly promising for lily breeding. INTRODUCTION Since 1960 about 7,000 lily cultivars have been registered (Leslie, 1982). Active lily breeding work started in Japan and the United States between the 1920’s and 1940’s. During the past 25 years it has been predominantly carried out in the Netherlands. The acreage increased from 100 hectares in 1970 till almost 4,800 in 2000. Breeding was in the beginning mainly focused on Asiatic hybrids, the 1980’s the Oriental hybrids became popular. Wide interspecific lily cultivars, with the LA-hybrids first, became commercial in the 1990’s. Meanwhile it is clear that more groups including OT (Oriental x trumpet), LO (longiflorum x Oriental) and OA’s (Oriental x Asiatic) will follow in the near future. The development of methods for overcoming fertilization barriers was essential in order to achieve the successes in interspecific lily breeding. A range of techniques were investigated and applied, besides pollination methods (cut-style, grafted style) to overcome pre-fertilization barriers and a number of techniques for post-fertilization barriers are needed. Embryo-rescue methods (ovary-slice, ovule and embryo culture) are needed to circumvent the problems with the embryo-endosperm development, mitotic and meiotic polyploidization (chromosome doubling using oryzalin or colchicine or by 2n-gametes) could overcome the F1-sterility which most frequently occurs. The last method to prove and speed up introgression of characters in interspecific hybrids is the so-called GISH (Genomic in situ hybridization), which can distinguish the parental genomes of Proc. IX Intl. Symp. on Flower Bulbs Eds.: H. Okubo, W.B. Miller and G.A. Chastagner Acta Hort. 673, ISHS 2005 100 interspecific hybrids. Mitotic polyploidization or somatic chromosome doubling results in tetraploid interspecific hybrids with recovered fertility, however, no homoeologous recombination can be seen in subsequent progenies (Lim et al., 2000). On the other hand, meiotic polyploidization has shown high frequencies of homoeologous recombination in lily hybrids (Lim et al., 2001, 2003; Van Tuyl et al., 2003), which is needed for introgression of characters. Therefore the search of 2n-gamete producing genotypes is essential for breeding with sterile interspecific hybrids. In this paper four different types of interspecific lily hybrids, for which the occurrence of 2n-gametes are described and the probable approaches for using the sexual polyploids are discussed. MATERIAL AND METHODS Plant Material The plant material used originated from the PRI-breeding programme (Van Tuyl et al., 1989, 1991, 2000) as shown in Table 1, except the Lilium auratum x L. henryi hybrid which was created by Asano (1981). Chromosome Preparation and In Situ Hybridization Chromosomes were prepared by squashing fixed cells onto clean microscope slides. The method was modified from that of Karlov et al. (1998). Well squashed preparations were dehydrated in a graded ethanol series after taking off cover slip and air dried. Genomic in situ hybridization was performed according to Lim et al. (2001). RESULTS AND DISCUSSION From Fig. 1, the crossing polygon of the genus Lilium, it is clear that many interspecific cross combinations were successful. In all these cases through mitotic polyploidization fertile tetraploids were produced. It was proved however that these tetraploids were not suitable for inducing any intergenomic recombination, which is essential for introgression characters (Lim et al., 2000). Therefore 2n-gametes producing genotypes are needed. This phenomenon is, however, rare among Lilium interspecific hybrids. During a period of 25 years in lily in 5 different types of hybrids 2n-gametes producing genotypes were found. Hybrids between ‘Enchantment’ x L. pumilum, an intra sectional hybrid, appeared not to be fertile like almost all intersectional hybrids, but they produce both n and 2n pollen (Van Tuyl et al., 1989). Using the Lilium auratum x L. henryi hybrid obtained by Yoshito Asano (1977), we produced a range of triploid Oriental-henryi hybrids in the early eighties. Now, 20 years later, using the GISH-technique, we could demonstrate that in many of these hybrids genetic recombination between the L. henryi and L. auratum chromosomes took place during meiosis of F1 hybrid. In Table 3, GISH-results are presented for a number of hybrids we obtained in 1982, 1983 and 1985 using ‘Journey’s End’, ‘Stargazer’, ‘Dominique’ and ‘Darling’ as female in crosses with F1 hybrid of L. auratum × L. henryi (Van Tuyl et al., 2002). Through a similar approach, we produced a large number of F1 hybrids between L. longiflorum x Asiatic hybrids and selected for 2n gamete producing genotypes (Lim et al., 2000, 2001). These were successfully back crossed to the parental species and obtained the BC1, triploid ALA progenies. GISH analyses of the ALA genotypes showed considerable frequencies of intergenomic recombination between the chromosomes of longiflorum and Asiatic hybrids. Despite having an odd polyploid number (3x), some of the ALA genotypes were successfully used as parents in crosses with both 2x and 4x parents and produced a large number of near diploid as well as near pentaploid progenies. From GISH analyses, of triploid and aneuploid progenies it was proved that whole genomes, individual chromosomes as well as recombinant chromosomes were transferred to the BC1 progenies. More recently, we have produced F1 hybrids between Oriental and Asiatic hybrids (OA) and selected 2n gamete forming genotypes. Using these, we have produced a large