Introduction
Moth orchids have elegant flowers with good longevity and are a promising floral plant with a huge market potential and value, as indicated by their prime status among potted floral plants (Been 2010). The majority of the moth orchids cultivated in South Korea consisted of Phalaenopsis cultivar developed through artificial crossing among Phalaenopsis and Doriteanopsis cultivar developed through breeding between Doritis pulcherrima and Phalaenopsis. As moth orchids are primarily tropical or thermophilic floral plants, South Korean farmers incur high cost of heating during cultivation in the winter season. Therefore, to develop a new moth orchid cultivar that adapts well to the lower temperature climate of S. Korea is important. Wind orchid flowers are indigenous to the southern islands of S. Korea and can survive throughout the winter season in the wild, and their 2 important types include Sedirea japonicum Linden & Rchb. f. and Neofinetia falcata (Lee 2011).
S. japonicum was first reported by the German botanist Reichenbach (1793-1879), and it was originally designated with the botanical name Aerides japonicum Linden & Rchb. f. However, it has since then been separated from the Aerides genus and assigned to the current Sedirea genus (Arditti 1977). S. japonicum is an evergreen epiphyte that grows on trees and rocks and coniferous forests found in the Bijarim of Jeju Island, located 150 m above the sea level, as well as in the South Jeolla Province and South Sea islands. Its roots, leaves, and flowers are the subjects of admiration. The roots are well-developed and the stem has 2–5 leaves alternatively arranged on both the sides of the stem. Possessing a thick mesophyll, the oblong-shaped leaves have a length of 7- 10 cm and a width of 1.5-2.5 cm. The flower has a light-green background color with red patterns, and 4-10 flowers blossom on the peduncle of a 5-15 cm size between late July and mid- August. Its blooming period spans approximately 20-30 days, and it has a high floricultural value due to its elegant fragrance (Kim and Lee 1979).
N. falcata leaves are narrower than those of S. japonicum, and the petals are long and thin, with persimmon-like fragrance that is subtle and less intense than that of S. japonicum. While the plant is popular among consumers for its fragrance and wintering capacity due to its psychrophilic nature; it is disadvantages in that the flower is short-lived and the flower color is plain white (Hur 2006). On the other hand, moth orchid flowers stay in the bloom for a long time (2-6 months), and exist in a variety of flower colors and shapes, although the flowers are thermophilic in nature and generally no fragrance.
In the present study, efforts were done to develop a new orchids via intergeneric hybridization of wind orchids and moth orchids, that is psychrophilic and has fragrance, intergeneric crossings of S. japonicum and of N. falcata with moth orchids were conducted. From these, the intergeneric hybrids were cultivated and superior lines were selected. Furthermore, in order to identify the morphological and genetic characteristics of the hybrid specimens, comparative analysis of the morphological traits and chromosomal characteristics was performed.Phalaenopsis × S. japonicum, 20 S. japonicum × Doritaenopsis, 37 Doritaenopsis × S. japonicum, 4 Phalaenopsis × N. falcata, and 5 N. falcata × Phalaenopsis.
Materials and Methods
Plant materials
The moth orchids used for intergeneric crossbreeding included approximately 89 species collected from the Joseph Wu orchid in Taiwan and the University of Hawaii in USA. The 62 strains of wild wind orchids used in intergeneric hybridization included S. japonicum and N. falcata purchased from the Agricultural Technology Center of Geoje-City in S. korea and S. japonicum from the Tsinghua Bio-one Seed Company.
Artificial crossing
With respect to the artificial crossbreeding to develop intergeneric hybrids of wind orchids and moth orchids, a total of 160 crossbreeds were generated in 2009. The crossbreeds consisted of 29 S. japonicum × Phalaenopsis, 65 Phalaenopsis × S. japonicum, 20 S. japonicum × Doritaenopsis, 37 Doritaenopsis × S. japonicum, 4 Phalaenopsis × N. falcata, and 5 N. falcata × Phalaenopsis.
Intergeneric hybrid development and selection of elite lines
After performing artificial crossing, formation of seed pods was examined. In case seed pods were formed, aseptic propagation on the Hyponex medium was performed to acquire the intergeneric hybrids. After propagating the seeds, the seed germination rate was schematically investigated and the germinated seedlings were subjected to flask cultivations, from which the specimens with complete root and shoot formation were acclimated to the greenhouse environment for 2 weeks before transplantation. The development of the seedling for intergeneric hybrids from 12 intergeneric cross combinations 890 hybrid seedling were obtained. Among the intergeneric hybrids that had bloomed, 2 superior lines (819- 3B and K9256) were selected based on the floral morphology, floral arrangement, and the number of flowers.
Analysis of morphological traits and chromosomal characteristics of intergeneric hybrids
Among the intergeneric hybrids acquired from intergeneric crossings in 2009, 2 specimens (819-3B and K9256) from 2 combinations (N. falcata × Phalaenopsis equestris and D. pulcherrima var. champonenis × S. japonicum) that exhibited normal blooming were compared for their inflorescence and floral shape, along with their morphology and pollinia formation mechanism. In order to observe their chromosomes, pollinia, depending upon the location and the size of the buds, were extracted from inflorescence that had not yet bloomed, fixated for 5 min in Carnoy’s solution, treated for 5 min with 1N HCl, squash stained in 1% aceto-orceine, and finally observed under a microscope(nikon) at 1,000 × magnification. Flow cytometry assay was performed using fresh leaves collected from the known parents of the crossbreeds and intergeneric hybrids; the leaves were mixed with 400 μL of nucleus extracting solution for approximately 30 s and then cut using a razor blade into small pieces and passed through a 30-μm filter. The filtrate was stained with 1.6 mL of 4,6-diamidino-2- phenylindole (DAPI) solution and subjected to ploidy analysis using a ploidy analyzer. Flow cytometry assay was performed at Kyungpook National University, Daegu, Korea.
Fertility of intergeneric hybrid
to acquire the progeny of the intergeneric hybrid specimens, back-crossing and self-crossing of 7 combinations with both the parents were performed. After the artificial crossing, observations were made regarding the fertilization status, pod formation, and fall-blooming phenomenon in the plants.
Result and Discussion
The morphological characteristics of intergeneric hybrids
From a total of 160 cross-combinations performed to generate intergeneric hybrids, 2 strains that were psychrophilic and showed superior floral shape and color were selected (819-3B and K9256). When the intergeneric hybrid (819-3B) obtained from the crossing of D. pulcherrima and S. japonicum began to bloom, floral characteristics were examined to compare the morphological traits of the progeny to those of the parents. As demonstrated in Fig. 1, the flower from D. pulcherrima was approximately 3 cm in size and was of pure white color. Its lip and lateral lobes were yellow, and the lateral sepals also had yellow tips. The 2 pollinia were attached to each other, which split into 2 strands to form a total of 4 pollinia. The flower of S. japonicum was of approximately 3 cm in size, of white color with a slight green tinge, and its lateral sepals displayed pink stripes with a unique characteristic pink spot at its prominent lip. The pollen had 2 pollinia and exhibited a round shape with a slightly visible lobe. The flower of intergeneric hybrid specimen from D. pulcherrima and S. japonicum cross was morphologically similar to its mother, D. pulcherrima. The color of the flower was also similar to that of D. pulcherrima, but its prominent lip showed a pink hue similar to that of S. japonicum. The pollinia of the intergeneric hybrid specimen showed similar traits as D. pulcherrima, but the polliniun size was smaller than that of the parents.
The morphological traits of intergeneric K9256 hybrid from N. falcata and P. equestris were also compared to those of the parent. As shown in Fig. 2, the flower of N. falcata had thin petals and sepals that were 2.5-cm long, with a thin and long spur spanning 8 cm and the color of the flower was white. The pollen had 2 spherical pollinia of approximately 1 mm size, with a slight lobe. In the case of the father plant, P. equestris, the flower was star-shaped and of approximately 2–2.8 cm size; the flower had a white background with pinkish center and a pink prominent lip. The pollen of the father plant had a sharp oval-shaped tip with a severe lobe, and each polonium was separated into 2 parts. The flower of intergeneric hybrid specimen had a floral shape and size intermediate of that of the parent plant. The flower color was adopted from that of the father plant, P. equestris, that is, it had a pink prominent lip and petals with white background and pinkish center portion. In addition, although the flower size was small, it possessed a spur that was characteristically similar to N. falcata flower. The pollinia size and shape of intergeneric hybrid specimen were intermediate of those of both the parents, and the extent of lobe was similar to that of P. equestris lobe.
Investigation of pollen mitosis in crossed parents and the hybrids
Investigation of the optimal period for pollen mitosis in the parents of D. pulcherrima × S. japonicum cross and F1 hybrids was conducted (Fig. 3 and Table 1).
As shown in Fig. 3, the inflorescence of D. pulcherrima was upright and showed a continuous blooming, indicative of indefinite inflorescence. The specimen used in this experiment possessed a continuous bloom of 10 flowers, and the buds of each flower were collected immediately after the bloom for examination. The examination revealed that the second bud from the first flower exhibited a high chromosome frequency and that the bud size used at that time was 6-7 mm. In S. japonicum, blooming began from the donor bud only after the inflorescence was developed to a certain level and the blooming was completed within a few days. Therefore, because it was difficult to determine the appropriate blooming period, inflorescence prior to blooming was selected and investigated based on the bud location. As a result, when the length of the inflorescence was approximately 10-15 cm and 10 buds had formed, pollen mitosis was observed in the second bud from the terminal bud and the size of the bud at that time was 8-9 mm. The intergeneric hybrid specimen from D. pulcherrima × S. japonicum exhibited a flowering habit similar to that of D. pulcherrima and showed a continuous blooming of more than 10 flowers. After the flowering had begun, the 6th bud from the topmost terminal flower was found to be most useful for chromosomal studies; at that time, the size of the bud was 8-9 mm (Table 1).
Fig. 4 and Table 2 show the results of investigation of the appropriate pollen mitosis period in both the parents of N. falcata × P. equestris cross and the F1 hybrids.
As the inflorescence of N. falcata tended to show simultaneous blooming of 4-6 flowers, the buds were collected based on their size and examined to reveal a bud size of 4- 5 mm, with the highest frequency of pollen mitosis. Although the inflorescence of P. equestris exhibited branching, based on the order of blooming, the second or third bud was found to be the most appropriate for chromosomal studies and the size of the buds was fond to be approximately 7 mm. The flowering habit of intergeneric hybrid specimen was similar to that of P. equestris and, considering the blooming order, the 3rd or 4th bud was found to the most useful for chromosomal observation and the size of the buds at that time were 8-9 mm (Fig. 4 and Table 2).
Ploidy analysis
Studies on the chromosomal behavior in the intergeneric hybrids require a significant amount of time and effort and, even in the case of ploidy analysis, microscopic examinations revealed erroneous results (Singh 1984). to examine the applicability of flow cytometry, contrast analysis was performed on the results from microscopic examination of chromosomes and flow cytometry. Table 3 showed the comparative results of the microscopic examination of chromosomes and flow cytometry in this study.
Flow cytometry was performed to test the ploidy of 819-3B intergeneric hybrid specimen (D. pulcherrima × S. japonicum), K9256 intergeneric hybrid specimen (N. falcata × P. equestris), and their parents. Fresh leaf tissue samples from the hybrids and their respective parents were collected. As shown in Table 3, investigation of the ploidy of both the parents used for intergeneric cross revealed them to be diploids; this observation was consistent with the chromosome count confirmed in pollen mitosis. However, in the case of F1 hybrids, pollen mitosis examination results were not consistent with the flow cytometry results. The results from flow cytometry showed mostly diploids, with only few triploids. Examination of the leaf tissue of the orchid by flow cytometry to test the ploidy revealed various changes in the ploidy between the tissues and specimens. However, ploidy testing with leaf tissues is extremely difficult (Lin 2001). With regard to the ploidy analysis of intergeneric crossing of orchids, the development of a more accurate measurement method that uses flow cytometry testing protocol in conjunction with microscopic examinations are required (Lee 2005).
Cytogenetic analysis of intergeneric hybrid
Fig. 5 showed the observational results of the pollen mitosis between the parents of the D. pulcherrima × S. japonicum cross and F1 hybrids. The majority of Phalaenopsis species had a chromosome count of 2n = 38 with a size range of 1.5- 3.5 μm (Arends 1970). In addition, the chromosome count of wind orchids was found to be mostly 2n = 38. Fig. 5a and 5b show the pollen mitosis of D. pulcherrima, while Fig. 5c showed the somatic mitosis of root tip cell.
Chromosome count of n = 19 was observed (Fig. 5), which confirmed diploidy. As indicated by Fig. 5c, when microscopic examination of root tip cell chromosome was performed, the number of chromosomes appeared to be approximately 50. Therefore, D. pulcherrima seems to be a tetra-ploid, although further analysis is needed to confirm the same. As seen in Fig. 5d-5f, the microscopic examination of S. japonicum pollen chromosome showed a normal pollen tetrad formation and a chromosome count of n = 19. Pollen mitosis of F1 hybrid from D. pulcherrima × S. japonicum was observed (Fig. 5g–5i), with a chromosome count of n = 20-40. Microscopic examination of pollen chromosomes revealed that both the parents exhibited a normal pollen tetrad formation. In the case of 819- 3B hybrid specimens, mutations among the specimens were severe and normal pollen tetrad formation was not observed. Moreover, the pollen chromosome count was 20-40 due to cell division from abnormal chromosome separation and exchange as well as from the occurrence of deletion and recombination between chromosomes. Because the female parent D. pulcherrima var. champonensis was a tetra-ploid, there is a high probability that the observations of the chromosome during intergeneric crossing may be extremely complex. Min and Tan (1996) reported that, in a study of Paphiopedilum pollen meiosis, abnormal spindle formation, delay in univalent formation, and abnormal chromosome separation may have contributed to mutations in the chromosome count, whereby, although the normal chromosome count is n = 19, a count of n = 5-22 was recorded. Despite the abnormalities in the chromosomal count, all the pollens were confirmed to be fertile.
Fig. 6 indicated the pollen mitosis micrograph of both the parents from N. falcata × P. equestris cross and F1 hybrids.
N. falcata (Fig. 6a-6c) exhibited a normal tetrad formation with a count of n = 19. P. equestris (Fig. 6d-6f) exhibited a count of n = 19 20, which is consistent with that reported by a previous study (Sagawa and Shoji 1968), and a normal tetrad. However, F1 hybrids were irregular with a count of n = 20-50 and the highest frequency of n = 30 (Fig. 6g-6i). Besides normal tetrad formation, dyad and micronucleus formations were also commonly observed (Fig. 6k and 61). A study on intergeneric crossing of cattleya by Silva-Stort (1984) reported irregular microspore genesis, resulting in the formation of not only tetrads but also of triads and dyads and even monads in some cases. Some studies also reported micronucleus, with all pollens being sterile.
Fertility of intergeneric hybrids
For fertility testing of intergenerically crossed F1 hybrids, back-cross was performed using intergeneric hybrid specimens and both the parents of D. pulcherrima and S. japonicum cross and hybrid specimens and both the parents of N. falcata and P. equestris. The results of this fertility testing are shown in Table 4.
A combination of S. japonicum as a cross-parent and an intergeneric hybrid from D. pulcherrima × S. japonicum as a pollen parent was used to perform 10 cross-breeding. From among these, 2 specimens showed pod formation. When S. japonicum was used as the paternal plant and D. pulcherrima × S. japonicum as the maternal plant, 4 pods were formed from 12 crosses. After fertilization, the success rate of back-crossing was approximately 30%. However, crosses with intergeneric hybrid from N. falcata and P. equestris did not result in any pod formation, despite numerous attempts, and falling of the flower was observed to occur much sooner than normal (Fig. 7).
Fig. 7a showed the result of sib cross 5 days after fertilization; at this time, all flowers had fallen, rendering the plant sterile. As Fig. 7b showed, in P. equestris × (N. falcata × P. equestris) cross combination, pod formation did not occur even at 10 days after pollination. As also indicated by the back-cross results of Fig. 7c and 7d, no pod formation was noted even at 25 and 30 days after pollination, resulting in the falling of all flowers. Based on these results, it was determined that intergeneric hybrid specimens from N. falcata and P. equestris are sterile. It was reported that sterility may be caused by micronucleus observed during the tetrad formation process, together with abnormal tetrad formation and mutation in the chromosome count. Therefore, further studies on the sterility of these F1 hybrids are required along with designing of measures to avoid development of such conditions.
When morphological and cellular genetic traits of intergeneric hybrid specimens selected for this study were examined, the progeny presented intermediate types of both the parents, confirming that they were definitely intermediate hybrids. The selected hybrid specimens in this study displayed psychrophilic and summer-flowering traits. Our results may provide helpful insight to the development of new varieties that are psychrophilic and flower in summer season as a new product or for use as a maternal line in the future research.
