Meiotic polyploidization in five different interspecific Lilium hybrids
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Presentation for the International Symposium on Flower bulbs april 2004, Japan / Meiotic polyploidization in five different interspecific Lilium hybrids Jaap M. van Tuyl, Rodrigo Barba-Gonzalez, Alex A. van Silfhout, Ki-Byung Lim & M.S. Ramanna Plant Research International, Wageningen University and Research Centre,P.O. Box 16, 6700 AA Wageningen, The NetherlandsFax: +31 317 418094 E-mail: jaap.vantuyl@wur.nlAbstractWith 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, investigated them for the occurrence of 2n gametes, sexual polyploid progenies were produced and they 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 inter sectional 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 were 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. Further more, 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. Keywords: 2n-gametes, homoeologous recombination, introgression, FDR, SDR, IMR INTRODUCTION 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. MATERIAL AND METHODSPlant 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). Table 1. Plant material selected for meiotic and for GISH research.
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. 1989). Using the Lilium auratum 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 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:
Literature Cited Asano Y. 1982. Chromosome association and pollen fertility in some interspecific hybrids of Lilium. Euphytica 31:121 128. Karlov G.I., Khrustaleva L.I., Lim K.B. and Van Tuyl J.M. 1999. Homoeologous recombination in 2n-gamete producing interspecific hybrids of Lilium (Liliaceae) studied by genomic in situ hybridization (GISH). Genome 42:681686. 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. Lim, K.B., Shen, T.M. and Van Tuyl J.M. 2004. Occurrence of numerically unreduced gametes in Lilium hybrids and its relevance for breeding. Breeding Science 54: in press. Van Tuyl J.M. 1989. Research on mitotic and meiotic polyploidization in lily breeding. Herbertia 45:97 103. Van Tuyl, J.M. and Van Holsteijn, H.M.C.1996. Lily breeding research in the Netherlands Acta Hortic 414: 35-45. Van Tuyl J.M., De Vries J.N., Bino R.J. and Kwakkenbos A.A.M. 1989. Identification of 2n-pollen producing interspecific hybrids of Lilium using flow cytometry. Cytologia 54: 737-745. Van Tuyl J.M. and De Jeu, M.J. 1997. Methods for overcoming interspecific crossing barriers. Chapter13 In: Sawhney and Shivanna (eds.), Pollen Biotechnology for Crop Production and Improvement, Cambridge University Press, 273-293. Van Tuyl J.M., Van Dien M.P., Van Creij M.G.M., Van Kleinwee T.C.M., Franken J. and Bino R.J. 1991. Application of in vitro pollination, ovary culture and embryo rescue for overcoming incongruity barriers in interspecific Lilium crosses. Plant Science 74:115–126. 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. Van Tuyl J.M., Maas I.W.G.M and Lim K.B. 2002. Introgression in interspecific hybrids of lily. Acta Hortic 570: 213-218.
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