Ki-Byung Lim & M.S. Ramanna
Since 1960 about 7.000 lily cultivars has 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 raised from 100 hectares in 1970 till almost 4800 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, LO and OA’s 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 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; Lim et al. 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.
Figure 1. Crossing polygon genus Lilium
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 Figure 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 type 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 inter sectional hybrids, but they produce both n and 2n pollen (Van Tuyl et al.
Using the Lilium auratum
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
(Van Tuyl et al.
Through a similar approach, we produced a large number of F1 hybrids between L. longiflorum
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 number of BC1, triploid, OOA and OAA progenies. GISH analyses of these triploids have demonstrated that there is considerable frequency of intergenomic recombination between the chromosomes of Oriental and Asiatic species.
In all the four inter sectional hybrids meiosis was highly irregular during meiosis and produced almost exclusively first division restitution (FDR) gametes. On the contrary, the intra sectional hybrid, ‘Enchantment’ x L. pumilum
had quite normal chromosome pairing and produced only second division restitution (SDR) gametes.
From the three instances described above the following conclusions can be made:
Through proper screening, it is possible to select genotypes of F1 hybrids of lilies that can produce fairly high frequencies of 2n gametes and these can be successfully used for generating BC progenies.
Unlike somatic chromosome doubling, use of 2n gametes can successfully produce intergenomic recombination. This facilitates the expression of recessive phenotypes in the BC1 progenies already and this could be a reason why cultivars have been selected by using the BC1 progenies produced in the case of both AH and LA hybrids.
GISH analysis of BC1 and subsequent progenies can provide valuable information for developing rational approaches for –breeding lilies more efficiently.
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Lim K.B., Ramanna M.S., De Jong J.H., Jacobsen E. and Van Tuyl J.M. 2001. Indeterminate meiotic restitution (IMR): a novel type of meiotic nuclear restitution mechanism detected in interspecific lily hybrids by GISH. Theor. Appl. Genet. 103:219-230.
Lim K.B., Chung J.D., Van Kronenburg B.C.E., Ramanna M.S., De Jong J.H. and Van Tuyl J.M. 2000. Introgression of Lilium rubellum Baker chromosomes into L. longiflorum Thunb.: a genome painting study of the F1 hybrid, BC1 and BC2 progenies. Chromosome Research 8:119-125.
Lim K.B., Ramanna M.S., Jacobsen E. and Van Tuyl J.M. 2003. Evaluation of BC2 progenies derived from 3 x 2 and 3 x 4 crosses of Lilium hybrids: a GISH analysis. Theor. Appl. Genet. 106:568-574.
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Van Tuyl J.M., Van Dijken A. Chi H.S., Lim K.B., Villemoes S. and Van Kronenburg B.C.E. 2000. Breakthroughs in interspecific hybridization of lily. Acta Hortic 508: 83-88. 247-252.
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