Interspecific Hybridization of Wild Gladiolus Species Using a Dendrogram Based on RAPD Analysis

Y. Takatsu, K. Suzuki, T. Yamada1, E. Inoue2, T. Gonai, M. Nogi and M. Kasumi Plant Biotechnology Institute Ibaraki Agricultural Center 319-0292, Ibaraki Japan

1Present address 2Present address National Institute of Floricultural Science School of Agriculture, Ibaraki University Tsukuba, 305-8519 Ami, Inashiki, 300-0393 Japan Japan

Keywords: gladiolus, wild species, dendrogram, interspecific hybridization

Abstract We have obtained information on relationships among wild gladiolus species through RAPD analysis (Takatsu et al., 2001b). Interspecific hybridization has been carried out based on a dendrogram and F1 seeds have been derived from seven reciprocal crosses. However matured seed was not obtained from the crosses using Gladiolus orchidiflorus as a female parent. We have made a segregation population (F2) derived from interspecific hybridization between G. tristis and G. gracilis for selection of a RAPD marker linked to a floral scent trait of the wild species G. gracilis. Floral scent of each plant was evaluated by sensory evaluation in the F2 plants and it was shown that a segregation ratio of scented plant and non-scented plant fit a 1:3 (χ2-value=0.09) ratio.

INTRODUCTION Modern gladiolus (Gladiolus x grandiflora hort.) is an important cut flower product in Ibaraki prefecture Japan, and novel characters (like floral scent, shape and so on) are required to increase consumption. The genus Gladiolus is classified in the family Iridaceae, and the current number of species in the genus is 255 (Goldblatt and Manning, 1998). Modern cultivars are considered to have been bred originally from only six to 12 species (Anderton and Park, 1989; Barnard, 1972; Imanishi, 1989), and considerable genetic potential exists to develop new cultivars of gladiolus using wild species. Especially, some species having a good scent should provide useful material for breeding of a scented modern cultivar. However, there is little information of genetic relationships in wild gladiolus for interspecific hybridization. Recently, Goldblatt and Manning (1998) proposed a phylogenetic tree for 163 southern African gladioli using morphological, geographic and ecological data. Although their study was unique and useful, we have not been able to obtain sufficient information, such as cross compatibility for interspecific hybridization among wild species. Takatsu et al. (2001b) have generated a dendrogram based on RAPD (randomly amplified polymorphic DNA) analysis, however the utility of this dendrogram in a breeding program of gladiolus has not been investigated. As a next step, we have tried interspecific crosses among wild species based on the dendrogram, and examined cross compatibility and hybridity of obtained F1 plants. In addition, a segregation analysis of the floral scent trait was carried out using F2 population derived from crosses between G. tristis and G. gracilis.

MATERIALS AND METHODS

Plant Materials Thirty-two wild gladiolus species of southern Africa origin (Fig. 1) were purchased

Proc. IXth Intl. Symp. on Flower Bulbs 475 Eds.: H. Okubo, W.B. Miller and G.A. Chastagner Acta Hort. 673, ISHS 2005 from Silverhill Seeds Co. (Cape Town, S.A). These species and one modern cultivar G. x grandiflora hort. ‘Traveler’ were used for RAPD analysis, and a dendrogram was generated from the data of RAPD analysis among 33 species (Takatsu et al., 2001b).

Interspecific Crosses Seven reciprocal interspecific crosses within or between clusters were made (Table 1). Parental plants were grown in a greenhouse maintained between 12-30°C under natural light conditions. Pollen was collected and stored at -20°C until pollination for 1-2 months as described by Takatsu et al. (2001a). Pollen viability was tested before pollination using the method of Heslop-Harrison and Heslop-Harrison (1970) with fluorescein diacetate. Matured F1 seeds obtained for the interspecific crosses were harvested 2 month after pollination, and were sown immediately. The germination rate of the obtained seeds was calculated as follows: (number of seedlings/number of seeds) x 100. Twelve months after sowing, seedlings with small bulblets were harvested as putative hybrids. Hybridity of F1 seedlings was confirmed by flow cytometric analysis, using the methods described in material of the High Resolution Kit for Plant DNA (Partec GmbH, Germany).

Segregation Analysis We chose G. tristis (cluster 3) and G. gracilis (cluster 2) as parent species having a different type of scent. G. tristis is a species scented strongly in the early evening (clove-like) having pale-yellow flower color and flowering time is September to November in southern Africa. G. gracilis is sweetly scented during the day (violet-like) having a pale-blue flower and the flowering time is mainly July and early August in the place of origin (Goldblatt and Manning, 1998; Takatsu et al., 2002). Segregating population (F2) was obtained by self pollination of an F1 plant derived from interspecific cross between these species. Some characteristics (days to flowering and floral scent) were examined in the population and compared with the parent species. Characteristic of floral scent in F2 plants was examined using the modified method of a sensory evaluation described by Morinaka et al. (2002).

RESULTS AND DISCUSSION

Interspecific Crosses We selected five parent species for the interspecific cross based on their interesting and unique flower color and floral scent traits (Table 1). G. gracilis and G. tenellus are in cluster 2, and G. uysiae, G. watermeyeri and G. orchidiflorus are in cluster 3 on the dendrogram (Fig. 1). Three reciprocal crosses between G. orchidiflorus x G. uysiae, G. orchidiflorus x G. watermeyeri and G. uysiae x G. watermeyeri were done in the same cluster, but they are not very close to each other on the dendrogram. Four reciprocal crosses between G. gracilis x G. orchidiflorus, G. gracilis x G. uysiae, G. gracilis x G. watermeyeri and G. tenellus x G. uysiae were ‘inter-cluster’ crosses (Fig. 1, Table 1). On crosses in the same cluster 3, we obtained mature seeds from three crosses of the six combinations, but seed was not obtained from three crosses of G. orchidiflorus x G. uysiae, G. orchidiflorus x G. watermeyeri and G. watermeyeri x G. uysiae. In the inter-cluster crosses, mature seeds were obtained from most combinations except for the cross G. orchidiflorus x G. gracilis. The germination rates of the seeds ranged from 10.2 to 67.7% (Table 1). Although the fluorescent intensity of F1 seedlings was confirmed by flow cytometric analysis, significant difference was not observed in F1 seedling and their parents (Table 3). Takatsu et al. (2002) suggested that it was possible to confirm a hybridity of F1 plant using flow cytometry, because fluorescent intensity was different depending on wild gladiolus species. However, fluorescent intensity was very close in the parent species used in this study. So, further investigations are needed to confirm the hybridity of F1 seedlings by morphological data or RAPD analysis. We have not been able to obtain seed from the combination using G. orchidiflorus as a female parent. The other wild gladiolus species used in this study are diploid plants

476 (2n=2x=30), but G. orchidiflorus is thought to be a triploid plant (Goldblatt and Manning, 1998). Takatsu et al. (2002) also showed that the species was triploid (2n=3x=45) by chromosome counting, but also reported that G. orchidiflorus produced pollen and a lot of seed was obtained by self-pollination. Although the reasons are not cleared why matured seed was not obtained in crosses using this species as a female parent, it might be that the ploidy level of this species affects cross compatibility. Matured seeds were obtained in 10 cross combinations, in spite of crosses in the same cluster or ‘inter-cluster’ crosses. This suggests that compatibility of interspecific crosses was not related to the position on the dendrogram of five species used in this study. From our results, we consider that it should not be difficult to produce interspecific hybrids of wild gladiolus species in a breeding program. Although we used only species that flower in the spring in this study, Takatsu et al. (2001a) have suggested that air temperature affected to pollen tube growth, fertility and seed set in interspecific hybridization of gladiolus. So we recommend that pollination be done at low temperatures (15-20°C).

Segregation Analysis Fifty-six plants were successfully obtained as a segregating population (F2) derived from crossing G. tristis x G. gracilis. Days to flowering is based on the days from planting of bulbs to flowering. This characteristic of F2 plants showed broad distribution, but the days were elongated in all of F2 plants compared with the parent species (Fig. 2). G. gracilis as a male parent and G. tristis as a female parent needed about 90 days and 150 days to flowering, respectively. Whereas F1 plant required about 220 days to flower and the days for F2 plants ranged from 160 to 210 days. Although this result suggests that days to flowering of F1 plants shows transgressive segregation and the characteristic is under the control of a quantitative gene, there is room for further investigations. Floral scent is one of the interesting traits in gladiolus breeding, and scented species will likely play important roles in the development of new modern cultivars. The scent of F2 plants was evaluated by sensory evaluation at 10:00 am in fine weather for two days to eliminate an alteration of strength of scent based on climate. F1 plants derived from G. tristis x G. gracilis do not have a sweetly scent during the day. However, 13 plants of 56 F2 population had a violet-like scent specified to G. gracilis in a segregating population and the segregation ratio of scented plants to unscented plants was 1:3 (χ2-value=0.09, Table 2). This suggests that the floral scent trait of G. gracilis might be under the control of a single recessive gene. We are going to screen a specific RAPD by a bulked segregant analysis using this F2 population to identify a DNA marker specified to scented plants.

ACKNOWLEDGEMENTS We thank Dr. Takeshi Motozu (Horticultural Research Institute, Ibaraki Agricultural Center) for his excellent advice. This work was supported by a grant for scientific research from the Ministry of Agriculture, Forestry and Fisheries, Japan (1997-2002).

Literature Cited Anderton, E.W. and Park, R. 1989. Growing gladioli. p.12-13. Timber Press, Oregon, USA. Barnard, T.T. 1972. On hybrids and hybridization. p.304-310. In: G.J. Lewis, A.A. Obermeyer and T.T. Barnard (eds.), Gladiolus, a revision of the South African species, J. S. Afr. Bot. Suppl. vol. 10. Goldblatt, P. and Manning, J. 1998. Gladiolus in Southern Africa. Fernwood Press, Vleaberg, S. Afr. Heslop-Harrison, J. and Heslop-Harrison, Y. 1970. Evaluation of pollen viability by enzymatically induced fluorescence; intracellular hydrolysis of fluorescein diacetate. Stain Technol. 45:115-120. Imanishi, H. 1989. Gladiolus. (in Japanese). p.1077-1080. In: T. Matsuo (ed.), Collected

477 data of plant genetic resources, Kodansha Scientific, Tokyo. Morinaka, Y., Takatsu, Y. and Hayashi, H. 2002. Sensory evaluation of floral scent in freesia (Freesia hybrida hort.) cultivars. (in Japanese with English summary). J. Jpn. Soc. Hort. Sci. 71:702-709. Takatsu, Y., Kasumi, M., Manabe, T., Hayashi, M., Inoue, E., Marubashi, W. and Niwa, M. 2001a. Temperature effects on interspecific hybridization between Gladiolus x grandiflora and G. tristis. HortScience 36:341-343. Takatsu, Y., Miyamoto, M., Inoue, E., Yamada, T., Manabe, T., Kasumi, M., Hayashi, M., Sakuma, F., Marubashi, W. and Niwa, M. 2001b. Interspecific hybridization among wild Gladiolus species of southern Africa based on randomly amplified polymorphic DNA markers. Scientia Hort. 91:339-348. Takatsu, Y., Manabe, T., Kasumi, M., Yamada, T., Aoki, R., Inoue, E., Morinaka, Y., Marubashi, W. and Hayashi, M. 2002. Evaluation of germplasm collection in wild gladiolus species of southern Africa. (in Japanese with English summary). Breed. Research 4:87-94.

Tables

Table 1. Results of seven reciprocal crosses using five wild gladiolus species in the same cluster, or in different cluster of dendrogram shown in Fig. 1.

Crossing1 Combinations No. of No. of Germination Female parent Male parent obtained obtained rate2 seeds seedlings (%) Crossing in G. orchidiflorus G. uysiae 0 --3 -- the same G. uysiae G. orchidiflorus 62 42 67.7 cluster 3 G. orchidiflorus G. watermeyeri 0 -- -- G. watermeyeri G. orchidiflorus 202 53 26.2

G. uysiae G. watermeyeri 39 23 58.9 G. watermeyeri G. uysiae 0 -- -- Inter-cluster G. gracilis G. orchidiflorus 19 4 21.1 crossing G. orchidiflorus G. gracilis 0 -- --

G. gracilis G. uysiae 38 7 18.4 G. uysiae G. gracilis 58 27 46.6

G. gracilis G. watermeyeri 461 47 10.2 G. watermeyeri G. gracilis 6 1 16.7

G. tenellus G. uysiae 38 7 18.4 G. uysiae G. tenellus 2 1 50.0 1 Crossing in the same cluster 3: Interspecific cross between wild species being in cluster 3 of dendrogram shown in Fig. 1. Inter-cluster crossing: Interspecific cross between wild species in different clusters of the dendrogram shown in Fig. 1. 2 Germination rate was calculated as follows: (number of seedlings/number of obtained seeds) x 100. 3 --: No data

478 Table 2. Segregation of scented plants and unscented plants among F2 population derived from interspecific hybridization between Gladiolus tristis and G. gracilis.

No. of scented plants No. of unscented plants Total χ2-value (1:3) 13 43 56 0.09 (P=0.75)

Table 3. Fluorescent intensity obtained by flow cytometric analysis among three wild gladiolus species and their F1 seedlings.

Materials of analysis1 Fluorescent intensity2 G. uysiae 110.1±1.8 G. uysiae x G. watermeyeri 111.1±1.1 G. watermeyeri 117.9±0.8 G. gracilis 108.3±1.4 G. gracilis x G. watermeyeri 105.9±1.5 G. watermeyeri 117.9±0.8 G. tenellus 116.2±1.9 G. tenellus x G. uysiae 108.5 G. uysiae 110.2±0.9 1 Each column shows the species used as a female parent, a male parent and their F1 seedling. 2 Mean±SD (n=3)

Figures

Fig. 1. Dendrogram generated from the data of 133 RAPD among 33 gladiolus species. These species are separated into 4 major clusters in this dendrogram described by Takatsu et al. (2001).

479

20

15

10 G.tristis F1 plant G.gracilis Number of plants of Number 5

0

0 60 -1 61-170 101-110 111-120 121-130 131-140 141-150 151 1 171-180 181-190 191-200 201-210 211-22

Days to flowering

Fig. 2. Distribution of days to flowering among F2 plants derived from interspecific hybridization between Gladiolus tristis and G. gracilis. White arrows indicate the days to flowering of parent species G. tristis and G. gracilis, and their F1 plant, respectively.

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