4. Directions of lily breeding research

4.1. Lily research at CPRO

At the Centre for Plant Breeding and Reproduction Research (CPRO-DLO, former IVT) in Wageningen lily breeding research was started 20 years ago. Results are presented in this paper and the most important publications are listed in the list of references. The last four years lily research increased enormously. This is especially due to the Urgency Programme for Bulb Diseases and Breeding Research. At this moment we have research projects on:

* Interspecific hybridization, mitotic and meiotic polyploidization and management of the cultivar and species collection (Keijzer et al.1988; Van Roggen et al. 1988; Van Tuyl 1985c, 1986, 1990; Van Tuyl et al. 1982, 1986, 1988ab, 1989abc, 1990, 1991ab, 1994, 1996).

* Breeding for Fusarium resistance (researchers: Huub Löffler & Dolf Straathof*; Löffler & Mouris, 1989, 1990; Löffler & Straathof, 1990)

* Tagging Fusarium resistance using molecular markers a project carried out in cooperation with the Test Centre for Ornamental Crops Ltd (Hillegom). In this project economical important characters (e.g. Fusarium resistance) are located on a genetic map with molecular markers, which can be detected easily in an early stage of development. This method can speed up the breeding process very much (Straathof et al. 1995).

* Breeding research on keeping quality of the lily and tulip flower (Van der Meulen, et al. 1996)

* Long term storage of bulb crops (Bonnier & Van Tuyl, 1996)

* Development of microspore culture for in vitro selection: the culture of isolated microspores via callus or embryos is a promising system for in vitro selection at the haploid level (Van den Bulk et al. 1991; Van den Bulk & Van Tuyl 1996)

* Pollen transformation: application of genetic manipulation in monocot plant species is hampered by the lack of transformation and regeneration procedures. A new method being investigated is the introduction of foreign DNA using particle gun ( Bino et al. 1991)

* Introduction of virus resistance in lilies by transformation of the virus coat protein, research carried out at the University of Leiden (researcher: Simon Langeveld)

* Protoplast technology: production of lily protoplasts, regeneration to whole plants, protoplast fusion (somatic hybridization) and transformation of protoplasts (Famelaer et al. 1996).

Several of these projects are carried out on the basis of 50% financing by the Dutch Commodity Board for Ornamental Horticultural Products (PVS) in the Urgency Program for Bulb diseases and Breeding research.

In this paper only some topics of this research, namely the interspecific hybridization and polyploidization work, are summarized.

4.2 Interspecific hybridization

4.2.1. Introduction

Within the genus Lilium the about 85 species can be classified into seven sections. The group of the Asiatic hybrid cultivars, which is derived from species of the section Sinomartagon is economically the most important group for cut flower production in the Netherlands. The lily assortment, however, might be considerably improved by exploiting economically important traits from other Lilium species. The species used in our interspecific hybridization programme (Van Tuyl et al. 1986, 1988b) were chosen on the basis of their respective characters: L. candidum (section Lilium) with pure whiteness, fragrance and low light and temperature tolerance; L. longiflorum (section Leucolirion) with forcing ability and growth vigour; L. henryi (section Archelirion) with resistance to virus diseases and bulb rot (caused by Fusarium oxysporum ) and L. pumilum with earliness and a bright red colour (section Sinomartagon). The reasons for using polyploidy in lily breeding are the larger flowers and the stronger stems of polyploid plants (especially important for forcing under low light conditions during the winter period, Van Tuyl et al., 1985). In interspecific hybridization the F1-sterility at the diploid level is restored tetraploid level. Tetraploids can be obtained by colchicine treatment of mitotic cells (mitotic polyploidization). In contrast to the mitotic, the meiotic or sexual polyploidization produces polyploids through the fusion of two gametes (pollen or eggs cells), one or both having the double number of chromosomes. Meiotic polyploidization has a great potential, it is a natural method with the following advantages above the colchicine treatment:

* no occurrence of chimeras,

* no decreased growth and fertility caused by the toxic effect of

colchicine,

* genotypic and environmental dependency and

* the outbreeding effect in contrast to mitotic polyploidization

where inbreeding takes place.

The following topics illustrate the research on interspecific hybridization:

The use of in vitro pollination techniques (4.2.2.), the development of the LA-hybrids (4.2.3.), results of crosses with 5 Lilium species (4.2.4.), mitotic polyploidization for overcoming F1-sterility (4.2.5.) and the analysis of ploidy chimeras using flow cytometry (4.2.6.) and meiotic polyploidization: the detection of 2n-gametes (4.2.7.)

4.2.2. In vitro pollination techniques in wide hybridization

The use of a complete and integrated in vitro system for pollination, fertilization and embryo rescue in lily was examined. By combining pollination techniques to overcome pre-fertilization barriers with in vitro methods to overcome post-fertilization barriers, both under fully controlled conditions, interspecific lily crosses could be made more efficiently. In vitro cut-style pollination and in vitro grafted style technique were developed and applied on various interspecific crosses using L. longiflorum, and both Asiatic and Oriental hybrids as parents. In addition, methods for ovary culture, ovary-slice culture and ovule culture were generated. Ovule swelling score in ovary culture was used to evaluate media effects on ovule development.

The optimal media and time of application (in DAP=days after pollination) developed for the in vitro methods are summarized in Table 3; MS is standard Murashige & Skoog medium (Van Tuyl et al. 1990, 1991a).

The research on new pollination techniques has been carried out in close cooperation with the Department of Plant Morphology and Cytology of the Agricultural University (Janson, 1990; Keijzer, et al.1988; Van Roggen et al.1988; Stals et al.1988).

4.2.3. The development of LA-hybrids

Within the genus Lilium L. longiflorum is one of the most interesting species carrying many characters required in horticulture. Parallel to the breeding programme with L. longiflorum aimed at improving bulb production under Dutch climate conditions (chapter 3), interspecific crosses were made at the IVT with other white lilies viz. L. candidum and the white asiatic hybrids 'Mont Blanc' and 'Whilito' (Van Tuyl et al.1988a). In the summer of 1980 9 cut-style pollinations produced 3 set pods using L. longiflorum 'White Europe' and 'Mont Blanc'. 42 Days after pollination 11 embryos were rescued by in vitro culture. In 1982 8 hybrids of this cross came into flower. General characteristics of these hybrids are: white or creamy white flower colour with fewer spots than 'Mont Blanc', 'floppy' flower size, little or no ornamental value, sterile pollen, and often dark green L. longiflorum foliage. The only hybrid with some ornamental value, which we called 'Loblanca' , large asiatic type flowers. In 1984 this hybrid was backcrossed to 'Mont Blanc' and L. longiflorum. The cross with 'Mont Blanc' produced 25 embryos. 'Lomonta' is one of these large flowered triploid, upfacing hybrids which came into flower in 1986. From bulbgrowth experiments it was demonstrated that these hybrids have a growth vigour comparable with those of L. longiflorum. Various clones from these crosses were released to the Dutch private breeding firms (Van Tuyl, 1987). Using colchicine a tetraploid 'Loblanca' was developed which showed complete restoration of the pollen fertility (see also 4.2.5.). Crosses with tetraploid F1-hybrids appeared to be more successful than similar ones at the diploid level. The latter crosses mainly produce sterile triploids. Meanwhile several Dutch breeding companies are very busy to develop from this material in combination with their own material commercial cultivars. This group of hybrids is called LA-hybrids because of their L. longiflorum and Asiatic origin and are characterized by large flower sizes, pastel flower colours and a striking growth vigour.

4.2.4. Recent results of crosses using five Lilium species

Using ovary- and ovule culture in combination with different pollination techniques wide interspecific crosses were made with several lily species (L. longiflorum, L. dauricum, L. henryi, L. candidum and L. concolor) and cultivars (f.e. the Asiatic hybrid 'Whilito')(Van Tuyl et al. 1991ab). A major part of the hybrids (more than 100) obtained after culturing more than 50.000 ovules flowered in 1991. Except two all in vitro cultured ovules, appeared to be hybrids. This can be determined visually rather easy. Remarkable was the large variation in flower shape, spotting and colour observed in the cross of L. longiflorum with L. dauricum. The white coloured L. longiflorum and the yellow coloured and heavy spotted L. dauricum produced a progeny of plants with a complete different spotting and flower colour. The flower colour ranged from almost white, via creme, light yellow, pink, bicoloured yellow-pink to dark purple and violet. The latter colours are virtually unknown within the Asiatic hybrid group. The hybrids originated from the cross of L. longiflorum and L. concolor all showed a light red flower colour and a flower size intermediate between both of the parents. Interesting combinations are those in which L. henryi is used as a parent because of the enormous vigour and the resistance of this species (for Fusarium, virus and Botrytis). For the first time several hybrids from the cross L. longiflorum with L. henryi came into flower. Although these five hybrids didn't show a perfect flower form, the vigour was remarkable and they were even partly fertile. The hybrids of L. henryi x L. candidum were also exceptional vigorous and demonstrated fully restored fertility after in vitro chromosome doubling.

In general wide interspecific hybrids in lily present sterility because of cytogenetical difficulties during meiosis. This sterility hampers further breeding with these hybrids. Because these problems were expected all hybrids were tested for pollen fertility. This was done using a vitality stain FDA and by germinating pollen on an artificial medium. All normal lily genotypes have a vitality between 60-100%. It is remarkable that about 9% of the plants exhibit a pollen fertility of more than 25%. When subsequently pollen fertility was tested by germinating the pollen on an artificial medium, the results were similar. Research (see 4.2.7.) showed that this fertility was caused by the occurrence of 2N-gametes. This means that the pollen grains formed have the double number of chromosomes. Further breeding with this material is possible when tetraploid material is available.

In Figure 1 a crossing polygon is presented of the genus Lilium. All successful crosses between different sections of the genus Lilium obtained at CPRO-DLO are included. In this figure the connection between the Asiatic, Aurelian and Oriental hybrid groups (ellipses) are shown by dotted lines. In successful crosses between species (small circles) of different sections (large circles) the arrow point towards the female parent. In most cases L. longiflorum has been used successfully as female parent.

The hybrids, obtained after intersectional crosses, are produced by using the cut style method in combination with embryo culture or with ovary-slice and ovule culture. It has not been proved that by using the grafted style method other combinations succeeded than after using the cut style method, but the number of hybrid embryos obtained per ovary increased, in some combinations, substantially with the GSM-method. However, a problem of the grafted style method is that the pollen tubes often are not able to enter the style of the mother ovary because of the inadequate attachment of the styles.

In this study reciprocal differences in crossing barriers are demonstrated. In the crosses with L. longiflorum this species was only successful when used as mother. Especially the Oriental x Asiatic hybrids, a combination of the two commercially important lily groups, are a break-through in lily breeding and a promise for the future. This combination appeared to be more difficult than other crosses. The percentage of hybrids per pollinated flower appeared to be very low and genotype dependent. In Table 5 the percentage ingrowth of pollen of one Asiatic father in 8 Oriental mothers are compared with the percentage hybrid plants (number of hybrids/number of pollinated flowers) obtained after cut style pollination and application of different embryo rescue methods of in total 228 ovaries. It can be concluded from this data that a high percentage of ingrowth is correlated with a higher number of hybrid plants.

From several interspecific combinations flowering plants have been obtained. The number of obtained plantlets and the number of these plants which flowered are listed in Table 4. The plants, especially the OA-hybrids, have not all flowered yet, so the number of flowering plants will increase.

The reported hybrids are partly described by others before (Asano 1980, Okazaki et al. 1994). The crosses between the species L. longiflorum x L. martagon var. cattaniae, L. longiflorum x L. monadelphum, L. longiflorum x L. canadense and L. henryi x L. candidum have not been reported before. Verification of hybrids, in an early stage when visual observation is not yet possible, was carried out using flow cytometry (Van Tuyl & Boon, 1996).

Restoration of F1-sterility by doubling the number of chromosomes was performed successfully in the L. henryi x L. candidum, L. longiflorum x Asiatic hybrids, L. longiflorum x L. candidum, L. longiflorum x L. concolor, L. longiflorum x L. henryi and L. longiflorum x L. dauricum using oryzalin. Using these tetraploids backcrossings were performed on Asiatic, Oriental and Longiflorum hybrids.

For the near future tetraploid Lilium-hybrids originated from a range of different genotypes (L. longiflorum, L. henryi, Asiatic hybrids, Oriental hybrids), which could not be combined up till now, open complete new and promising possibilities for innovating the lily assortment.

Figure 1: A crossing polygon of the genus Lilium included all successful crosses of genotypes between different sections of the genus Lilium developed at CPRO-DLO. In this figure the connection between the Asiatic, Aurelian and Oriental hybrid groups (large ellipses) are shown by dotted lines. In successful crosses between species (small circles) of different sections (large circles) the arrow point towards the female parent.

Abbreviations: A: Aurelian hybrids; AL: L. alexandrae; AM: L. amabile; AS: Asiatic hybrids; AU: L. auratum; BU: L. bulbiferum; CA: L. candidum; CAN: L. canadense; CE: L. cernuum; CH: L. chalcedonicum; CO: L. concolor; DAU: L. dauricum; DAV: L. davidii; DU: L. duchartrei; FO: L. formosanum; HA: L. hansonii; HE: L. henryi; JA: L. japonicum; LA: L. lankongense; LEI: L. leichtlinii; LO: L. longiflorum; MA: L. martagon; MI: L. michiganense; MO: L. monadelphum; NO: L. nobilissimum; O: Oriental hybrids; PU: L. pumilum; RE: L. regale; RU: L. rubellum; SP: L. speciosum; SU: L. sulphureum; TI: L. tigrinum; TS: L. tsingtauense; Test: L. x testaceum.

Table 4: Total number of in vitro plantlets and the number of plants which produced flowering plants obtained from several interspecific cross combinations, using ovary-slice culture and other embryo rescue techniques.

SUCCESSFUL COMBINATIONS

                                         NUMBER

                                                              plantlets flowering

L. longiflorum x Asiatic hybrid   >100              >100

L. longiflorum x L. candidum     >100                 28

L. longiflorum x L. concolor         21                     8

L. longiflorum x L. henryi             16                   15

L. longiflorum x L. dauricum     >100                  56

L. longiflorum x L. bulbiferum       3                     2

L. longiflorum x L. martagon          1                    1

L. longiflorum x L. canadense        1                    1

L. henryi x L. candidum                3                     2

L. longiflorum x Oriental hybrid    25                   15

Oriental x Asiatic hybrid           >600                > 200

4.2.5. Mitotic polyploidization for overcoming F1-fertility

Besides using 2n-gametes fertility can be restored by chromosome doubling. An important advantage of the developed in-vitro techniques is that arising sterility can be restored by in-vitro chromosome doubling. This can be done in the same year in which ovules or embryos are cultured. As a consequence a considerable speed up of the breeding process can be realized. In the experiments for this purpose colchicine and oryzalin was used.

Oryzalin is developed as a herbicide and is found to be a metaphase inhibitor, like colchicine. Oryzalin however is characterized by a lower toxicity than colchicine and can be applied in lower concentrations and must be considered as an alternative for colchicine (Verhoeven, et al.1990). In an experiment with several sterile interspecific hybrids 0.1% colchicine (0.1%) and oryzalin (0.01% and 0.005%) was employed. The regenerated bulblets were tested for their level of ploidy using flow cytometry (see 4.5). Of 92 regenerated lily plants 31 appeared to be polyploid. A high percentage is chimeric (di_tetra, di_octa, tetra_octa) probably because of juvenility of the material. Oryzalin appeared to be a less inhibiting regeneration than oryzalin, and also the number of polyploids was higher. Disadvantageous effects like growth abnormalities caused by mutation induction possibly do not occur using oryzalin. Oryzalin can be considered as an alternative for the very toxic colchicine. Meanwhile the first tetraploid L. henryi x L. candidum came into flower and showed a fully restored fertility.