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Article

Interspecific Crossing between Lilium hansonii Leichtlin and L. brownii var. colchesteri for the Breeding of New Lily Cultivars

by
Ji-Young Kim
1,†,
Ye-Su Song
1,2,3,†,
Jong-Kuk Na
4,* and
Jong-Hwa Kim
1,2,*
1
Department of Horticulture, Kangwon National University, Chuncheon 24341, Korea
2
Oriental Bio-Herb Research Institute, Kangwon National University, Chuncheon 24341, Korea
3
Department of Herbal Pharmacology, College of Korean Medicine, Kyunghee University, Seoul 02447, Korea
4
Department of Controlled Agriculture, Kangwon National University, Chuncheon 24341, Korea
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2022, 12(3), 621; https://doi.org/10.3390/agronomy12030621
Submission received: 18 January 2022 / Revised: 25 February 2022 / Accepted: 1 March 2022 / Published: 2 March 2022
Browse Figures
Versions Notes

Abstract

:
This study aimed to generate interspecific hybrids between two remote lily species, L. hansonii Leichtlin and L. brownii var. colchesteri. Reciprocal crosses were performed by conventional and cut-style pollination methods, but viable seeds were only obtained when L. hansonii was used as the female parent, indicating that unilateral incompatibility exists between the two species. In the case of immature seeds, embryos with 2~3 mm were carefully removed from testa for further in vitro culture, and they grew as normal plants. A total of 343 progenies was obtained from the crosses, and hybridity of the progenies were examined using the L9 marker, simple sequence repeat (SSR) marker, at the seedling stage and 92 were confirmed as F1 hybrids. Ploidy level of 76 F1 hybrid was examined and confirmed as diploid. F1 hybrids exhibited intermediate morphologies of the parent in outer tepal and leaf length, but flower shape and color were similar to those of L. hansonii. On the other hand, F1 hybrid plants showed increased flower spots, flower size, and bud numbers, which could be important signatures of the F1 hybrid. This study reports the first attempt to generate an interspecific hybrid between the two species, and therefore, our results from this study would be very informative for future lily breeding.

1. Introduction

Korea is one of the major wild lily habitats due to the ideal environment for Lilium prosperity [1]. There are 13 wild lilies native to South Korea, consisting of 11 wild lily species [2] and two varieties [3]. They are classified into two sections, Martagon and Sinomartagon [3].
L. hansonii, belonging to section Martagon, is one of Korean endemic wild lily species, found only in Uleung-Do [4], an island located in the East Sea, South Korea. On the other hand, L. brownii var. colchesteri is distributed in China and Korea and classified into section Archelirion [5] or section Leucolirion [6]. However, recent molecular studies using simple sequence repeat (SSR) and inter-retrotransposon amplified polymorphism (IRAP) markers revealed that L. brownii var. colchesteri is closely related to section Archelirion [7,8], supporting the classification by Comber [5]. There could be confusion between L. brownii var. colchesteri and L. brownii, but they have key differences in flower color, fragrance, and leaf shape. Nevertheless, both L. hansonii Leichtlin and L. brownii var. colchesteri have great potential as breeding materials to produce new lily cultivars.
L. hansonii is not only a lily species found in Uleung-Do but also L. lancifolium, L. amabile, and L. leichtlinii var. maximowiczii [9]. However, L. hansonii do not grow with other lilies likely because of its habitat under heavy shade with good drainage, where other lilies cannot grow well. The average bulb weight is around 20~25 g and the circumference is around 6.5~8.0 cm, respectively. The number of whorled leaves ranges from 9 to 10 from the first layers [9]. Flower buds are initiated in late fall inside the bulb and completed next spring when the plants sprout [10]. The flowering time of L. hansonii at Uleung-Do is from late May to early June [9] as early as that of L. concolor [11] and L. brownii var colchesteri (the author’s observation from a garden with germplasm collections, Kangwon National University, Chuncheon, Korea). Bulbs of L. hansonii have been used as a food in Uleung-Do from Choseon dynasty [12]. The characteristics of L. hansonii include early flowering [9], resistance to Fusarium [13], many flowers, and long vase life [14]. Due to such agricultural potentials, L. hansonii has been regarded as an invaluable breeding material.
L. brownii var. colchesteri has very special characteristics not found in other lilies. Its flower has a color transition from pale yellow to white during flowering and a sweet fragrance [15,16]. Additionally, L. brownii var. colchesteri has many other recommendable traits for cut and pot flowers. However, L. brownii var. colchesteri is very weak to the viruses including Cucumber mosaic virus, Lily mottle virus, and Lily symptomless virus, soil mites, and Fusarium [17], which may explain why this species is disappearing in Japan in recent years [15]. Unlike L. brownii var. colchesteri, L. brownii do not have a flower color change and scent [15]. Additionally, it has a different leaf shape: L. brownii has lanceolate leaves in equal length, but L. brownii var. colchesteri has broad leaves above or in the middle of the stem [6,18]. In this regard, L. brownii var. colchesteri is much more desirable for breeding material than L. brownii.
Lily breeding began a couple of hundred years ago, but the reliable breeding system started only 50 years ago. Most robust breeding has been conducted for Asiatic hybrids (known as the Division I group) within Lilium section Sinomartagon compared to interspecific hybrids among other sections. Although interspecific hybrids could show desirable distinct characteristics compared to the cultivars from the existing cultivars or species from the same section, the species from different sections are difficult to hybridize [14]. Many early cultivars of interspecific hybrids of L. × marhan and L. × dalhasonii were bred a long time ago in Europe by lily enthusiasts [19,20]. After developing various pollination and embryo rescue methods [21], intersectional crosses of L. longiflorm × L hasonii and Asiatic lily × L. hansonii have been successful [14]. In some cases, stigma pollination by mixed incongruous pollen is used to overcome the pre-fertilization barrier in lily breeding, and it performs better than cut-style pollination by single male pollen [22]. Recently the number of cultivars from interspecific hybrids has increased very rapidly [14] along with such improved pollination methods overcoming a bottleneck to obtain interspecific hybrids. Through such improved pollination techniques, cultivars from interspecific hybridization are rising rapidly, but there are still many species yet to be fully utilized for interspecific hybridization.
Prior to this study, intersectional crosses between L. hansonii and L. brownii var. colchesteri have not been reported. Here, we conducted reciprocal crosses between L. hansonii and L. brownii var. colchesteri for the first time to generate interspecific hybrids between L. hansonii into the L. brownii var. colchesteri that have invaluable horticultural traits. To obtain interspecific hybrids, several different pollination methods were applied and embryo culture was also applied to save immature seeds. Subsequently, F1 hybrid plants were selected using EST-SSR markers at early seedling stages.

2. Materials and Methods

2.1. Plant Materials and Pollination

Three lines of L. hansonii, donated as bulbs from a farmer in Uleung-Do, were used as parent lines, designated as P1, P2, and P3. One line of L. brownii var. colchesteri was used as another parent line (Accession no. 282,746 at Korean Agricultural Culture Collection of RDA, Jeonju, Korea), designated as P4. The bulbs were planted in a field with a red clay soil under the shade without protection in Chuncheon, Gangwon-Do, Korea (GPS: N 37°99′ E 127°69′).
Flowering of L. hansonii began in early June and lasted until late June, while that of L. brownii var. colchesteri began in the middle of June and lasted until early July in the field in Hwacheon-Gun, Korea (GPS: N 38°02′ E 127°72′). Therefore, reciprocal crosses were performed during mid to late June in 2013 when both species were flowering. Crosses were carried out using four different pollination methods, normal stigma pollination, cut-style pollination [23], and both normal stigma and cut-style pollinations using mixed incongruous pollens. Crosses were performed for 10~20 flowers for each method. For normal stigma pollinations, stigmas of emasculated plants were capped with aluminum foil, then pollinated one day after flowering of adjacent flowers, and recapped. Cut-style pollinations were conducted one day after flowering, in which style was removed at 1~3 mm above the ovary and the remaining style was cut vertically for opening to insert pollens [24]. For pollination by mixed incongruous pollens, pollens were mixed in equal parts of pollens from L. hansonii and L. brownii var. colchesteri before crossing.

2.2. In Vitro Culture of Mature Seeds and Immature Embryos

Mature fruits were harvested 60 days after pollination. All seeds were separated from fruits and washed by shaking for 3 min in distilled water with a few drops of Tween 20 and sterilized with 70% ethanol for 1 min and followed by 2% sodium hypochlorite solution for 15 min. Then, the seeds were rinsed in distilled water three times and placed in MS medium supplemented with 30 g/L sucrose, 0.8 g/L agarose, and 0.1 g/L activated charcoal. The seeds were germinated in a growth room with a condition of 12 h day/night at 23 °C. For DNA extraction, the leaves were collected from one-year-old seedlings cultured in vitro. As for immature seeds, embryo within testa were cultured for three weeks when the size of the embryo reached about 2~3 mm. Then, embryos were carefully removed and transferred to culture further on fresh MS medium.

2.3. Screening F1 Hybrid Using SSR Markers

Genomic DNA was isolated from young leaves using the DNeasy Plant Maxi kit (Qiagen, Germantown, MD, USA) according to the manufacturer’s instructions. DNA quantity was adjusted to 50 ng/µL and used for PCR analysis. Simple sequence repeat (EST-SSR) markers were used for hybrid selection at the early seedling stage. EST-SSR markers used in this study were developed previously from expressed sequence tags (EST) of the genus Lilium [8,25].
PCR analyses were conducted for all 343 progenies in a 25 µL reaction as described previously [8]. The amplified products were electrophoresed in a 6% denaturing polyacrylamide gel in a conventional PAGE system for 2 h at 1.8 kV or in a LiCor 4300 automatic electrophoresis system for 4 h.

2.4. Analyses of Morphology and Ploidy Level of F1 Hybrid

For phenotypic analysis of the F1 hybrid, qualitative and quantitative characters were examined for 10 F1 hybrid plants grown in a field of germplasm collections at Kangwon National University, Chuncheon, Korea (GPS: N 37°87′ E 127°74′). Qualitative characters including flower color, spots on flower, flower direction, scent, and leaf arrangement were examined. Quantitative characters included plant height, flowering time, flower diameters, and number of flower buds. Statistical analysis of quantitative traits was performed using one-way ANOVA imbedded in standalone Jamovi analysis tool.
To examine the ploidy level, the parent and 76 hybrid plants were analyzed by flow cytometry (Partec PA-1, Gorlits, Germany) using a method described by Kim, J.H., et al. [26]. DNA content was determined as C-values based on that of the Allium cepa (2C, approximately 33.5 pg) as a reference [27]. Previously, the 3C-value of triploid L. lancifolium was reported as 105.36 pg [28], and the 2C-value of diploid L. hansonii was reported as 101.02 pg [29]. In this study, the C-value of triploid L. lancifolium was also analyzed to compare and examine the possibility of the occurrence of the triploid F1 hybrid.

3. Results and Discussion

3.1. Generation of F1 Hybrid by Reciprocal Crosses of L. hansonii × L. brownii var. colchesteri

To obtain F1 hybrid lilies between L. hansonii and L. brownii var. colchesteri, reciprocal crosses were carried out using four different pollination methods. Average viable seed number from normal stigma pollination of L. hansonii (LH) × L. brownii var. colchesteri (LB) was 19 viable seeds per a capsule, whereas cut-style pollination failed to produce any fruit. In pollinations using mixed incongruous pollens, normal stigma pollinations produced 44 viable seeds per capsule, whereas cut-style pollination produced only six viable seeds from four fruits (Table 1). The crosses of L. hansonii × L. brownii var. colchesteri with normal stigma pollinations resulted in a total of 117 progenies. The crosses of L. hansonii × L. brownii var. colchesteri with mixed incongruous pollens produced 220 progenies from stigma pollinations and 6 from cut-style pollinations (Table 1). From all successful crosses, a total of 343 progenies were obtained. Fully developed fruit was about 3 cm in diameter and round as seen in the fruits of many other wild lilies (Figure 1A). The vertical section showed the fruits with full of seeds in various sizes (Figure 1B,C). However, mature seeds were obtained only from the center or upper part of the fruit. Ovules below the center of the fruit were immature or not fertilized. In the case of stigma pollination using mix incongruous pollens, percentages of viable seeds versus non-viable seeds were 35.5% versus 64.5%, respectively.
Intriguingly, progenies were obtained only when L. hansonii was used as the female parent (Table 1). It is known that both self- and cross-incompatibility are very common in Lilium species. We also found that unilateral incompatibility exists between L. hansonii and L. brownii var. colchesteri, in which it occurred when L. brownii var. colchesteri was used as the female parent in both normal stigma and cut-style pollinations as shown in Table 1. Stigma pollinations of L. brownii var. colchesteri × L. hansonii did not produce any fruit very likely due to unilateral incompatibility. Previously, it was reported that unilateral incompatibility was observed in a cross of L. formosanum × L. brownii var. colchesteri, in which the cross was only successful when L. brownii var. colchesteri was used as the male parent [15] as observed in this study. Unilateral incompatibility was also observed in crossing other lily species. An interspecific cross of L. longiflorum ‘Gelria’ × L. cernuum did not show any problem in pollinations, but the cross of L. cernuum × L. longiflorum ‘Gelria’ failed to produce any fruits [24]. The same phenomenon was also observed in the crosses of L. hansonii × L. leichtlinii, L. speciosum × L. leichtlinii, and L. auratum × L. henryi [30]. Since unilateral incompatibility is a common phenomenon in crosses between interspecific lilies [30], various factors have to be considered for successful production of interspecific hybrids such as pollination methods, necessity of embryo rescue, and the determination of the crossing direction between the two species used for breeding.
The success of crosses between self-compatible species and self-incompatible species typically depends on which species serves as female parent. In such cases, self-compatible species must be used as a female parent for successful pollinations [31]. Lily breeding has been hindered by self-incompatibility and incongruity barriers. In such cases, crosses may not produce any seeds, which led scientists to attempt various methods to solve the problem. Early studies were focused on the identification of proper methods to overcome pollination problems in intra- or interspecific crosses [21,32]. Such methods include the application of growth regulators, pollination after heating the style, cut-style pollination, and pollination with a pollen mixture [32]. Mixed pollen (incongruous pollen and congruous pollens) can be used to overcome pre-fertilization barriers in lily breeding because pollen incongruous to the female pistil can have fertility when they are used with mixtures with other compatible pollen.

3.2. In Vitro Culture to Increase Viability of F1 Hybrid Seeds

In lilies, the cross between two remote species is often accompanied by pre- and post-fertilization barriers. To overcome such problems, various pollination methods have been developed as described above. Cut-style pollination is one of the methods, which is normally applied with embryo rescue through in vitro culture [30]. This method was successful in interspecific crosses of L. longiflorum ‘Gelria’ × L. cernuum [24]. Post-fertilization incompatibility is often solved by in vitro culture of rescued embryos. Additionally, in vitro culture can increase the viability of immature seeds, which is applied in this study. Mature and immature seeds from 60-day-old fruits were sterilized (Figure 2A) and cultured in MS medium to increase viability of progenies (Figure 2). In the case of immature seeds, embryos inside testa were cultured until embryos reached a proper size to remove and transfer to new MS medium because many embryos did not grow well inside testa. Immature embryos grew to normal seedlings in a 3-month in vitro culture (Figure 2B) as did mature seeds (Figure 2C). Some immature seeds had very immature embryos with linear shape, but they developed well to normal young plantlets after in vitro culture (Figure 2D). Morphological variations were observed among F1 seedlings (Figure 2E), which could be due to temporal phenotypic changes caused by the inadequate in vitro conditions because most of seedlings grew very normally after being transplanted to soil.
As described above, the cut-style pollination method is often used to overcome pre-fertilization incompatibility in lily crosses. In this study, the cut-style pollination method was also applied along with normal stigma pollination because it is unknown whether the two species are compatible or not in pollinations. However, the cut-style pollination method was not successful by producing only fruits with very few viable seeds (Table 1). A further in-depth study would be necessary to understand why the cut-style pollination did not work well in the present study.

3.3. Screening F1 Hybrid Using SSR Markers

Developing new ornamental crops can be achieved more efficiently with the integration of conventional breeding based on phenotypic assessment and molecular marker-assisted breeding [33]. Phenotypic traits have long been used as key characteristics to confirm hybridity of F1 plants. Such traits include the morphology of the flower, pubescent time, and seed coat color. However, morphological confirmation of hybridity of F1 plants takes quite a long time, which could be shortened by molecular marker-assisted selection at the early seedling stage. Additionally, molecular markers can be used to eliminate unwanted self-pollinated progenies at the early developing stage, which can help to reduce the cost and labor required for plant breeding. Molecular markers have been used frequently to confirm F1 hybrids from intra- or interspecific crosses [34]. Among various molecular markers, SSR markers are the most common markers for such purposes due to their high reproducibility and usability at any stage of plant development [33]. Because it takes at least three years for lily progenies to become suitable for phenotyping, it would be better to remove undesirable self-pollinated progenies at the earliest time to save labor and time.
Previously, we developed 34 informative SSR markers from Lilium expressed sequence tags (ESTs) from the NCBI database to analyze phylogenetic relationships among wild lily species in Korea, and they showed high polymorphism information content (PIC) value [8,25]. Among them, 14 SSR markers including L9, eL61, and eL17 were first tested for parent lines, female parents (P1~P3), and a male parent (P4) to examine whether they could produce distinct PCR amplicons in parent lines. From the test, it was found that three L. hansonii lines had likely significant genetic variations. In case of L9 SSR marker, each one of three L. hansonii lines exhibited different PCR amplicons (Figure 3). The eL17 marker showed the same amplicons in P1 and P3 but not in the P2 line (Supplementary Figure S1). On the other hand, the eL61 marker exhibited the same amplicons in P2 and P3 but not in P1.
Among the tested 14 SSR markers, the L9 marker was found to be suitable for F1 hybrid selection and used for the confirmation of the hybridity (Figure 3) because this marker could be used as a reference for the source information among three female parents, P1~P3. Using the L9 marker, a total of 92 plants were confirmed as F1 hybrid plants among 343 progenies (Table 1). Among 117 progenies obtained from the crosses by conventional stigma pollination, 31 plants (26.5%) were confirmed as F1 hybrid plants. On the other hand, 61 plants (27.0%) were confirmed as F1 hybrid plants among 226 progenies from the crosses using mixed incongruous pollens (Table 1). Seed yield rate was higher in the crosses using an incongruous pollen mixture than that of conventional stigma pollination using pollens from one male parent, but the yield rate of F1 hybrid seeds did not show a difference between two pollination methods (Table 1).

3.4. Flower Characteristics and Ploidy Level of F1 Hybrids

To examine phenotypes of the F1 hybrid, qualitative and quantitative characteristics were investigated (Table 2 and Table 3). Prior to in-depth investigation, 10 F1 hybrids were surveyed for morphological variations, but significant variations were not observed among them. Therefore, the 10 F1 hybrids were used to examine qualitative and quantitative traits. As for qualitative traits, the flower shape of F1 hybrid was similar to that of L. hansonii (Figure 4). Flowers of the F1 hybrid was pale yellow and had many spots compared to that of L. hansonii (Figure 4). Stigma color was intermediate as brownish-yellow, and pollens were light brown compared to those of both parents (Table 2). Leaf arrangement of L. hansonii and L. brownii var. colchesteri is whorl and scattered, and F1 hybrid showed scattered leaf arrangement as that of L. brownii var. colchesteri. L. hansonii has no scent, but L. brownii var. chochesteri has a strong sweet fragrance. It was very desirable for F1 hybrid to have such a good fragrance, but the scent of the F1 hybrid was weaker than that of L. brownii var. chochesteri (Table 2).
As for quantitative traits, flowering time of the F1 hybrid was around June 23 between those of both parents, but it did not differ significantly. F1 hybrids exhibited intermediate morphologies of the parent in length and width of outer tepal and leaf length. On the other hand, F1 hybrids exhibited bigger flower size and higher bud numbers compared to both parents. The flower size of F1 hybrids was 4.5~10.8 cm bigger compared to 12.6 cm of L. brownii var. colchesteri and 6.3 cm of L. hansonii. Bud numbers of the F1 hybrid were 12.1, which was about 6 more than both parents (Table 3). Height of the F1 hybrid was smaller than both parents as 60.2 cm (Table 3). Further analysis on the characteristics of F1 hybrid is in progress, which would give more details of F1 hybrids.
Ploidy level was examined for 76 F1 hybrids by flowcytomeric determination of DNA content using Allium cepa as a reference and L. lancifolium as a reference for triploid lily (Supplementary Figure S2). As a result, all tested F1 hybrids were confirmed as diploid with a 2C-value of ~100 pg and a triploid F1 hybrid was not found.

4. Conclusions

Interspecific crosses would provide great opportunities to generate new lily cultivars between two remote species that have not been used previously. L. hansonii Leichtlin and L. brownii var. colchesteri belong to different sections and have great agricultural traits, but the interspecific cross between the two species has not been attempted. This study aimed to make F1 hybrid through a reciprocal cross between the two species and obtained 343 progenies from crosses of L. hansonii × L. brownii var. colchesteri. However, no viable seeds were obtained from crosses of L. brownii var. colchesteri × L. hansonii. Marker-assisted selection using the L9 SSR marker was applied to screen F1 hybrids at the early developmental stage, and 92 F1 hybrids were confirmed as hybrid among 343 progenies. Seventy-six F1 hybrids were examined for ploidy level to check the potential occurrence of triploid, but all were confirmed as diploids. F1 hybrids exhibited bigger flower size with increased spots and produced significantly higher number of buds than both parents, revealing that the F1 hybrid of L. hansonii × L. brownii var. colchesteri could be very desirable as a new addition to lily cultivars. However, the height of F1 hybrids was shorter than both parents, which may be undesirable for commercial lily cultivar used for cut flowers, but it would be fine for home gardens and potted plants. We are trying to select more desirable candidates for further breeding. Nevertheless, this study is the first report of the interspecific cross between the two lily species and would be very useful and informative for future lily breeding.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy12030621/s1, Figure S1: Amplicons generated by eL17 and eL61 SSR markers. Figure S2: Flowcytometric determination of DNA content of interspecific F1 hybrids between L. hansonii and L. brownii.

Author Contributions

J.-Y.K. and Y.-S.S. conducted the experiments and analyzed data. J.-K.N. and J.-H.K. designed the experiments. J.-Y.K., Y.-S.S., J.-K.N. and J.-H.K. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This study was carried out with the support of the GSP Project No. 213007-05-5-SBM10, the Ministry of Agriculture, Food and Rural Affairs, Korea.

Data Availability Statement

Not applicable.

Acknowledgments

We appreciate the farmer who provided L. hansonii bulbs for this study. Also, we thank Nam-Soo Kim for giving us helpful assistant.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Lucidos, J.G.; Ryu, K.B.; Younis, A.; Kim, C.-K.; Hwang, Y.-J.; Son, B.-G.; Lim, K.-B. Different day and night temperature responses in Lilium hansonii in relation to growth and flower development. Hortic. Environ. Biotechnol. 2013, 54, 405–411. [Google Scholar] [CrossRef]
  2. Lighty, R.W. The lilies of Korea. In The Lily Yearbook; Royal Horticultural Society: London, UK, 1969; Volume 31, pp. 31–39. [Google Scholar]
  3. Lee, C.S.; Kim, S.-C.; Yeau, S.H.; Lee, N.S. Major lineages of the genus Lilium (Liliaceae) based on nrDNA ITS sequences, with special emphasis on the Korean species. J. Plant Biol. 2011, 54, 159–171. [Google Scholar] [CrossRef]
  4. Jeong, J.H.; Kwon, S.T. Variations of morphological characteristics of Lilium hansonii related with protein and isozyme bands. Acta Hortic. 1996, 414, 145–150. [Google Scholar] [CrossRef]
  5. Comber, H.F. A new classification of the genus Lilium. In The Lily Yearbook; Royal Horticultural Society: London, UK, 1949; Volume 13, pp. 86–105. [Google Scholar]
  6. Wilson, E.H. The Lilies of Eastern Asia: A monograph; Dulau & Company: London, UK, 1925; p. 110. [Google Scholar]
  7. Lee, S.-I.; Kim, J.-H.; Park, K.-C.; Kim, N.-S. LTR-retrotransposons and inter-retrotransposon amplified polymorphism (IRAP) analysis in Lilium species. Genetica 2015, 143, 343–352. [Google Scholar] [CrossRef] [PubMed]
  8. Lee, S.-I.; Park, K.-C.; Song, Y.-S.; Son, J.-H.; Kwon, S.-J.; Na, J.-K.; Kim, J.-H.; Kim, N.-S. Development of expressed sequence tag derived-simple sequence repeats in the genus Lilium. Genes Genom. 2011, 33, 727–733. [Google Scholar] [CrossRef]
  9. Roh, S.M.; Yeam, D.Y.; Kim, Y.J. Native bulb materials in wild and their production for the cultivation as a floricultural crop I, Survey and Bulb Production. J. Korean Soc. Hortic. Sci. 1978, 19, 129–146. [Google Scholar]
  10. Ohkawa, K.; Kano, A.; Nukaya, A. Time of flower bud differentiation in asiatic hybrid lilies. Acta Hortic. 1990, 266, 211–220. [Google Scholar] [CrossRef]
  11. Wilson, C.G. The parade of the lilies in 1948. In The Lily Yearbook; Royal Horticultural Society: London, UK, 1949; Volume 2, pp. 126–128. [Google Scholar]
  12. Ministry of Korean Home Affairs. Korean Island Information Book; Ministry of Home Affairs: Seoul, Korea, 1985; p. 1372. [Google Scholar]
  13. Ogilvie, L. Recent advances in our knowledge of lily diseases. In The Lily Yearbook; Royal Horticultural Society: London, UK, 1947; Volume 11, pp. 87–92. [Google Scholar]
  14. Lim, K.B.; Barba Gonzalez, R.; Zhou, S.; Ramanna, M.S.; van Tuyl, J.M. Interspecific hybridization in Lily (Lilium): Taxonomic and commercial aspects of using species hybrids in breeding. In Floriculture, Ornamental and Plant Biotechnology: Advances and Topical Issues; Kagawa University: Takamatsu, Japan, 2007; Volume 5, pp. 138–145. [Google Scholar]
  15. Okubo, H.; Hiramatsu, M.; Masuda, J.I.; Sakazono, S. New insight into Lilium brownii var. chochesteri. Floricult. Ornament. Biol. 2012, 6, 44–52. [Google Scholar]
  16. Kim, J.Y.; Kim, J.H. Color shifting cultivar ‘Jeolseigain’ of Lilium x formolongi Hort. from interspecific crosses with L. brownii var. colchesteri. Flower Res. J. 2017, 25, 86–90. [Google Scholar] [CrossRef]
  17. Masuda, J.I.; Thien, N.Q.; Hai, N.T.L.; Hiramatsu, M.; Takeshita, M.; Kim, J.H.; Nakamura, M.; Iwai, H.; Okubo, H. Production of virus-free bulblets by meristematic tip culture with antiviral chemical in Lilium brownii var. colchesteri. Hortic. J. 2011, 80, 469–474. [Google Scholar] [CrossRef] [Green Version]
  18. Wallace, R.W.; Wallace, R.W. Hybrid lilies. In The Lily Yearbook; Royal Horticultural Society: London, UK, 1932; Volume 1, pp. 42–52. [Google Scholar]
  19. Gilman, R. Listing of Registered and Some Unregistered Martagon Hybrids; Royal Horticultural Society: London, UK, 1993; Volume 46, pp. 84–90. [Google Scholar]
  20. Pfeiffer, N.E. Martagon-Hansonii hybrids brought up-to-date. In The Lily Yearbook; Royal Horticultural Society: London, UK, 1959; Volume 12, pp. 57–61. [Google Scholar]
  21. Van Creij, M.G.M.; Kerckhoffs, D.M.F.J.; van Tuyl, J.M. Application of four pollination techniques and of hormone treatment for bypassing interspecific crossing barriers in Lilium L. In Proceedings of the XIX International Symposium on Improvement of Ornamental Plants, Angers, France, 27 July 1998; pp. 267–276. [Google Scholar]
  22. Proscevicius, J.; Ranceliene, V.; Kleizaite, V. Application of mixed incongruous pollen for interspecific crosses of lilies. Floric. Ornam. Biotechnol. 2012, 6, 89–93. [Google Scholar]
  23. Asano, Y. Overcoming interspecific hybrid sterility in Lilium. J. Jpn. Soc. Hortic. Sci. 1982, 51, 75–81. [Google Scholar] [CrossRef] [Green Version]
  24. Kim, Y.J.; Park, S.M.; Kim, J.H. Application of in vitro culture methods for overcoming cross-incompatibility in interspecific crosses between L. longiflorum and L. cernuum. Korean J. Hortic. Sci. 2001, 19, 373–377. [Google Scholar]
  25. Lee, S.-I.; Kim, N.-S.; Kim, J.H.; Son, J.-H.; Park, K.-C. SSR Primer for Screening Race or Line of Lilium spp. Longiflorum Section and Use Thereof. Patent No. 1012713300000, 29 May 2013. [Google Scholar]
  26. Kim, J.H.; Truong, N.X.; Song, Y.-S.; Kim, N.-S. Natural triploid Lilium leichtlinii var. maximowiczii populations in Korea. Plant Species Biol. 2016, 31, 98–106. [Google Scholar] [CrossRef]
  27. Bennett, M.D.; Leitch, I.J. Nuclear DNA amounts in angiosperms: Progress, Problems and Prospects. Ann. Bot. 2005, 95, 45–90. [Google Scholar] [CrossRef] [Green Version]
  28. Nguyen, T.X.; Lee, S.I.; Rai, R.; Kim, N.S.; Kim, J.H. Ribosomal DNA locus variation and REMAP analysis of the diploid and triploid complexes of Lilium lancifolium. Genome 2016, 59, 551–564. [Google Scholar] [CrossRef]
  29. Du, Y.P.; Bi, Y.; Zhang, M.F.; Yang, F.P.; Jia, G.X.; Zhang, X.H. Genome size diversity in Lilium (Liliaceae) is correlated with karyotype and environmental traits. Front. Plant Sci. 2017, 8, 1303. [Google Scholar] [CrossRef] [Green Version]
  30. Marasek-Ciolakowska, A.; Nishikawa, T.; Shea, D.J.; Okazaki, K. Breeding of lilies and tulips-Interspecific hybridization and genetic background. Breed. Sci. 2018, 68, 35–52. [Google Scholar] [CrossRef] [Green Version]
  31. Onus, A.N.; Pickersgill, B. Unilateral incompatibility in Capsicum (Solanaceae): Occurrence and Taxonomic Distribution. Ann. Bot. 2004, 94, 289–295. [Google Scholar] [CrossRef] [Green Version]
  32. Van Tuyl, J.M.; Straathof, T.P.; Bino, R.J.; Kwakkenbos, A.A.M. Effect of three pollination methods on embryo development and seedset in intra- and interspecific crosses between seven Lilium species. Sex. Plant Reprod. 1988, 1, 119–123. [Google Scholar] [CrossRef]
  33. Patella, A.; Palumbo, F.; Galla, G.; Barcaccia, G. The molecular determination of hybridity and homozygosity estimates in breeding populations of lettuce (Lactuca sativa L.). Genes 2019, 10, 916. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  34. Hameed, A.; Saleem, M.Y.; Akhtar, K.P.; Shoaib, M.; Iqbal, Q.; Asghar, M. Molecular confirmation of intraspecific tomato (Solanum lycopersicum) hybrids and their evaluation against late blight and Cucumber mosaic virus. Mol. Biotechnol. 2017, 59, 234–240. [Google Scholar] [CrossRef] [PubMed]
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Figure 1. Morphology of fruits from the cross of L. hansonii × L. brownii var. colchesteri. (A): Fruits. (B): Fruit cut in half. (C): Potential F1 seeds from one fruit.
Figure 1. Morphology of fruits from the cross of L. hansonii × L. brownii var. colchesteri. (A): Fruits. (B): Fruit cut in half. (C): Potential F1 seeds from one fruit.
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Figure 2. In vitro culture of mature and immature seeds from the cross of L. hansonii × L. brownii var. colchesteri. (A). F1 seed sterilization. (B). Immature embryo removed from testa (left) and after a 3-month culture of embryos (right). Scale bar represents only the size of the immature embryo (left). (C). Fully developed seed (left) and germinated seedling after a 3-month in vitro culture (right). (D). Four different growth images of an immature seed. (E). Morphological variation in F1 seedlings during in vitro culture.
Figure 2. In vitro culture of mature and immature seeds from the cross of L. hansonii × L. brownii var. colchesteri. (A). F1 seed sterilization. (B). Immature embryo removed from testa (left) and after a 3-month culture of embryos (right). Scale bar represents only the size of the immature embryo (left). (C). Fully developed seed (left) and germinated seedling after a 3-month in vitro culture (right). (D). Four different growth images of an immature seed. (E). Morphological variation in F1 seedlings during in vitro culture.
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Figure 3. Marker-assisted selection of F1 hybrids from the crosses of L. hansonii × L. brownii var. colchesteri using the L9 SSR marker. P1, P2, and P3 indicate three L. hansonii lines used as the female parent and P4 indicates L. brownii var. colchesteri used as the male parent. The letter “H” indicates F1 hybrids from the cross between L. hansonii × L. brownii var. colchesteri. Green arrows indicate common alleles found in three L. hansonii lines (P1, P2, and P3). Blue arrows indicate specific alleles found in P3 L. hansonii. Red arrows represent a specific allele in L. brownii var. colchesteri, P4.
Figure 3. Marker-assisted selection of F1 hybrids from the crosses of L. hansonii × L. brownii var. colchesteri using the L9 SSR marker. P1, P2, and P3 indicate three L. hansonii lines used as the female parent and P4 indicates L. brownii var. colchesteri used as the male parent. The letter “H” indicates F1 hybrids from the cross between L. hansonii × L. brownii var. colchesteri. Green arrows indicate common alleles found in three L. hansonii lines (P1, P2, and P3). Blue arrows indicate specific alleles found in P3 L. hansonii. Red arrows represent a specific allele in L. brownii var. colchesteri, P4.
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Figure 4. Flower colors and spots of L. hansonii, L. brownii var. colchesteri, and their F1 hybrid. L. hansonii with yellow-orange flower and few spots (left), F1 with yellow flower with numerous spots, and L. brownii var. colchesteri with white to brown transition flower without spots.
Figure 4. Flower colors and spots of L. hansonii, L. brownii var. colchesteri, and their F1 hybrid. L. hansonii with yellow-orange flower and few spots (left), F1 with yellow flower with numerous spots, and L. brownii var. colchesteri with white to brown transition flower without spots.
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Table
Table 1. Summary of interspecific crossing between L. hansonii and L. brownii var. colchesteri.
Table 1. Summary of interspecific crossing between L. hansonii and L. brownii var. colchesteri.
Crossing
Combinations
(♀ × ♂)		Average Number of Viable Seeds per Fruit (Mature Fruit/Flowers Pollinated)Number of ProgeniesNumber of Hybrids Confirmed by SSR Marker
Stigma PollinationCut-Style PollinationStigma/Cut-Style Pollination
LH × LB19 ± 13.5 (7/10)0 (0/10)117/031
LH × mix (LH + LB)44 ± 11.6 (5/10)1.5 ± 0.6 (4/10)220/661
LB × LH0 (0/20)0 (0/20)0/00
LB × mix (LH + LB)0 (0/20)0 (0/20)0/00
Total 337/692
LH: L. hansonii; LB: L. brownii var. colchesteri; mix (LH + LB): a mixture of pollens from both L. hansonii and L. brownii var. colchesteri in equal ratio. Average numbers are represented with standard errors, and the fractions in parentheses indicate a number of mature fruits with viable seeds among pollinated flowers.
Table
Table 2. Comparison of qualitative characters between parent and F1 hybrid plants of L. hansonii (LH) × L. brownii var. colchesteri (LB).
Table 2. Comparison of qualitative characters between parent and F1 hybrid plants of L. hansonii (LH) × L. brownii var. colchesteri (LB).
SpeciesFlower Color(RHS) zStigmaColorPollenColorSpotFlowerDirectionScentLeafArrangement
LHYellow-orange (17B)YellowBrownFewDownNoneWhorl
F1Yellow (11D)Brownish-yellowLight-brownManySideWeakScattered
LBWhite/brown (N/A) YLight-brownBrownNoneDownStrongScattered
z RHS denotes Royal Horticultural Society color chart. Y Not applicable. N = 10.
Table
Table 3. Comparison of quantitative characters between parent and F1 hybrid plants of L. hansonii (LH) × L. brownii var. colchesteri (LB) and parent.
Table 3. Comparison of quantitative characters between parent and F1 hybrid plants of L. hansonii (LH) × L. brownii var. colchesteri (LB) and parent.
SpeciesFloweringTimePlantHeight (cm)Flower Diameter (cm)Numberof BudsOuter TepalLeafLength (cm)
Length (cm)Width (cm)
LHJune 21 ± 6 a z86.4 ± 5.7 b6.3 ± 0.5 b6.2 ± 2.3 a3.1 ± 0.3 a1.2 ± 0.3 a12.8 ± 2.7 a
F1June 23 ± 5 a60.2 ± 8.3 a17.1 ± 0.8 a12.1 ± 2.4 b6.2 ± 0.9 b3.1 ± 0.5 b10.7 ± 2.1 a
LBJune 25 ± 9 a107 ± 18 c12.6 ± 2.1 c5.7 ± 1.4 a16.4 ± 1.9 c4.8 ± 0.8 c10.3 ± 0.5 a
z Mean ± SD (n = 10). The same letter in the column denotes no significant difference based on Tukey post-hoc test at p ≤ 0.05.
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Kim, J.-Y.; Song, Y.-S.; Na, J.-K.; Kim, J.-H. Interspecific Crossing between Lilium hansonii Leichtlin and L. brownii var. colchesteri for the Breeding of New Lily Cultivars. Agronomy 2022, 12, 621. https://doi.org/10.3390/agronomy12030621

AMA Style

Kim J-Y, Song Y-S, Na J-K, Kim J-H. Interspecific Crossing between Lilium hansonii Leichtlin and L. brownii var. colchesteri for the Breeding of New Lily Cultivars. Agronomy. 2022; 12(3):621. https://doi.org/10.3390/agronomy12030621

Chicago/Turabian Style

Kim, Ji-Young, Ye-Su Song, Jong-Kuk Na, and Jong-Hwa Kim. 2022. "Interspecific Crossing between Lilium hansonii Leichtlin and L. brownii var. colchesteri for the Breeding of New Lily Cultivars" Agronomy 12, no. 3: 621. https://doi.org/10.3390/agronomy12030621

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