Phalaenopsis is a monopodial epiphytic orchid genus with a unique and attractive flower morphology. It is comprised of more than 70 species distributed in the subtropical and tropical countries of Southeast Asia (Christenson 2001, Cribb & Schuiteman 2012, Yuan et al. 2021). In addition to its long-lasting flowers, it has a wide variety of flower shapes, sizes, and colors (Chen et al. 2011). These features make Phalaenopsis one of the most popular and economically important pots for cut flowers on the horticultural market. In Asia alone, it is estimated that 84 million pots of Phalaenopsis are sold each year, accounting for 60 million pots from China, 14 million from Japan, and 10 million from other Southeast Asian countries (Hsu et al. 2018).

Owing to the increasing demand for commercial production, orchid breeders are striving to produce new cultivars with novel traits, such as improved flower morphology, color, and fragrance (Griesbach 2002, Hsu et al. 2018). Cultivars can be chosen as parents for breeding programs if they possess desirable qualities that can be incorporated to improve existing cultivars (Yuan et al. 2021). Most Phalaenopsis grown in Korea and approximately 60% of those grown in China have red flowers (Vo et al. 2019). Recently, there has been an increasing interest in plants with white, pink, especially in yellow colors with red stripes and/or spots (Hsu et al. 2018). Approximately 98 varieties have been reported in publications and listed in the Korean Seed & Variety Service (KSVS 2019). However, yellow cultivars are uncommon, and limited information has been reported.

Phalaenopsis cultivars consist of a wide range of chromosome numbers, with 2n=38, 57, 76-114 (Aoyama 1993). In the history of Phalaenopsis breeding, hybrids with enhanced quantitative and qualitative traits have been produced through interspecific hybridization between sections or subgenera. This may cause changes in the structure and number of chromosomes (Lai et al. 2005, Lee et al. 2020, Rieseberg et al. 1996, Shaked et al. 2001). Hybridization involving species with similar genome sizes, chromosome lengths, and numbers is usually expected to produce progeny with chromosomes that pair and divide normally (Kao et al. 2001, Lin et al. 2005). In contrast, hybridization between species possessing different chromosome sizes, genome sizes, or ploidy levels has often resulted in sterile progenies or poor chromosome pairing, which are problems often encountered by Phalaenopsis breeders (Chen et al. 2011, Lin et al. 2005). For instance, commercial cultivars of yellow Phalaenopsis have been obtained by crossing diploid species of the subgenus Polychilos. These possess larger chromosomes and a bimodal karyotype (Lee et al. 2017), with tetraploid standard-type cultivars. This hybridization leads to the production of sterile triploid progeny (Lee & Chung 2021).

Chromosomes that do not pair at all (univalent), pair only partially, or pair in groups (multivalents) may lead to the loss of genetic material (Bolanos-Villegas et al. 2008). The lack of crucial DNA sequences caused by chromosome loss in hybrids may hamper basic metabolic processes, which can eventually lead to a failure to produce fertile pollen (Dafni & Firmage 2000, Levin 2002). Therefore, choosing the correct parents for hybridization is imperative for successful breeding programs and for collecting pollen from desirable parents. Pollen viability is an important parameter of pollen quality (Dafni & Firmage 2000) which is directly proportional to fertilization success (Martins et al. 2010). Several staining tests for assessing pollen viability can be conducted using Alexander’s dye (Alexander 1969), fluorescein diacetate (Heslop-Harrison & Heslop-Harrison 1970), fluoro chromatic dye (FCR) (van der Walt & Littlejohn 1996), lactophenol cotton blue (LPCB) (Bellusci et al. 2010), 2,3,5-triphenyl tetrazolium chloride (TTC) (Abdelgadir et al. 2012), or thiazolyl blue (MTT) (Khatun & Flowers 1995) stain. In addition, in vitro germination using the medium proposed by Brewbaker and Kwack (1963) can be performed to assess pollen viability.

Determining the chromosome number, analyzing meiotic behavior and post-meiotic products, and assessing pollen viability can aid in the reproductive study of Phalaenopsis cultivars. Therefore, this study aimed to conduct a cytogenetic assessment, which included chromosome evaluation, sporad analysis, and pollen viability tests on two Phalaenopsis yellow cultivars, namely Phalaenopsis ‘Fuller’s Sunset’ and Phalaenopsis ‘Geumgongju’. The data gathered in the present study has provided findings that inform some of the basic knowledge required for breeding yellow Phalaenopsis orchids.

Plant materials and sample collection

Young roots were harvested from P. ‘Fuller’s Sunset’ (Fig. 1a) and P. ‘Geumgongju’ (Fig. 1b) provided by Kangsan Orchids, Busan, Korea. Roots were treated with 2 mM 8-hydroxyquinoline for 5 h at 25℃ to arrest cells at metaphase. They were then fixed in Carnoy’s solution (3:1 ethanol-acetic acid) overnight before being stored in 70% ethanol until use for mitotic chromosome preparation.

Fig. 1. Plant and flower characteristics of Phalaenopsis ‘Fuller’s Sunset’ (a) and Phalaenopsis ‘Geumgongju’ (b).

Pollinia from P. ‘Fuller’s Sunset’ and P. ‘Geumgongju’ were collected at five different flowering stages to evaluate their pollen viability. The flower development stages were defined as follows: (1) flower fully opened, (2) one day after flower fully opened, (3) two days after flower fully opened, (4) three days after flower fully opened, and (5) five days after flower fully opened.

Pollen viability by TTC staining

Pollinia viability was assessed by staining with TTC (Sigma Aldrich, USA) (Abdelgadir et al. 2012, Khatun & Flowers 1995, Mattson et al. 1947). Fresh pollinia were placed in a 1.5 mL centrifuge tube containing 0.5% TTC aqueous solution and incubated for 24 h at 25±1℃ in the dark. A 0.5% TTC aqueous solution was prepared by dissolving TTC (0.5 g) in a 50 mM sodium phosphate buffer solution with a pH of 7.4. The stained pollinia were rinsed with distilled water to remove any additional stains and were examined under a stereo microscope (SZ 51, Olympus, Japan). The area of red-stained pollinia was considered viable, whereas the portion of unstained pollinia was considered non-viable.

In vitro pollen germination

In vitro pollen germination was conducted as described (Yuan et al. 2018) with some modifications. Pollinia were dipped in 70% ethanol for surface sterilization for approximately 10 s and cultured in a germination medium containing 10% sucrose, 20 mg l⁻¹ boric acid, and 5% plant agar at 5.8 pH. The pollinia were incubated at 25±1℃ for each storage condition and kept in the dark for 20 d. The pollens were carefully squashed in 1 µg mL⁻¹ 4’,6-diamidino-2-phenylindole (DAPI) solution as evenly as possible, and pollen tube growth was visualized under a fluorescence microscope at 100×magnification. Approximately 100 pollen grains for each flower stage and storage condition were observed. Pollen grains were considered to have germinated when the pollen tube was elongated to twice the pollen grain size. No pollen tube growth indicates no germination.

Sporad Analysis

Sporad analysis was conducted as described (Bolanos-Villegas et al. 2008) with minor modifications. Pollinia from full-sized flower buds and during early anthesis were fixed in 3:1 Carnoy’s solution for 24 h, hydrolyzed in 1 N HCl for 1 h at 60℃, rinsed thrice with distilled water, and stained with 4',6-diamidino-2-phenylindole (DAPI). The materials used for observation were examined under a fluorescence microscope at 600×magnification. At least 100 pollen mother cells (PMCs) were categorized according to the number of sporads at the end of division II. Tetrads with four cells of uniform size were considered normal post-meiotic products. Tetrads with micronuclei were considered abnormal.

Chromosome analysis

A conventional cytogenetic method was used to evaluate the chromosomes of yellow Phalaenopsis cultivars. The results showed that P. ‘Fuller’s Sunset’ (Fig. 2a) and P. ‘Geumgongju’ (Fig. 2b) had hypotetraploid chromosomes possessing 2n=4x-1= 75 and 2n=4x-2=74, respectively. Both cultivars also had asymmetrical chromosomes, indicating a mixture of small and medium-to-large chromosomes. They also possess different numbers of large chromosomes in their chromosome compositions.

Fig. 2. DAPI-stained chromosomes of P. ‘Fuller’s Sunset’ (a) with 2n=4x-1=75 and P. ‘Geumgongju’ (b) with 2n=4x-2=74. Scale bar= 10 µm.

Pollen viability evaluation by staining and in vitro germination

The freshly collected pollinia of P. ‘Fuller’s Sunset’ and P. ‘Geumgongju’ were stained with TTC solution to evaluate their pollen viability during five different flowering stages (Figs. 3a, 3b). The stained areas on the pollinia corresponded to different degrees of viability, with the yellow or unstained areas considered non-viable or having low viability. In P. ‘Fuller’s Sunset’, results showed prominent viability in the first two flowering stages as shown by the heavy TTC staining. Partially stained pollinia could be observed on the more aged pollinia in the later flowering stages of this cultivar. Meanwhile, all pollinia of P. ‘Geumgongju’ from the five different flowering stages remained yellow or unstained, indicating that the pollinia were non-viable or had low viability.

Fig. 3. Pollen viability of P. ‘Fuller’s Sunset’ (a) and P. ‘Geumgongju’ (b) as evaluated by TTC staining at different flowering stages. Flower developmental stages are defined as: (1) flower fully open, (2) one day after flowering, (3) two days after flowering, (4) three days after flowering, and (5) five days after flowering.

In addition to pollen staining, in vitro germination was performed to assess pollen viability. The results confirmed the viability of P. ‘Fuller’s Sunset’ (Fig. 4a) because it showed pollen tube growth in all flowering stages after 10 d of incubation. However, in P. ‘Geumgongju’ (Fig. 4b), no pollen tube growth was observed at any stage. The results of the staining and germination tests on both cultivars were consistent and confirmed the viability of their pollinia at different flowering stages.

Fig. 4. Pollen viability of P. ‘Fuller’s Sunset’ (a) and P. ‘Geumgongju’ (b) evaluated through in vitro pollen tube germination. Pollen tube growth could be observed in P. ‘Fuller’s Sunset’ while no pollen tubes developed in P. ‘Geumgongju’. Arrows indicate the growth of pollen tubes.

Sporad analysis

Sporadic analysis showed differences between the post-meiotic products of the two Phalaenopsis cultivars. PMCs found in P. ‘Fuller’s Sunset’ (Figs. 5a, 5b) were 67% normal tetrads while 33% showed an abnormality, particularly sporads containing micronucleus. In contrast, results showed that 80% of the observed PMCs in P. ‘Geumgongju’ (Figs. 5c, 5d) were abnormal sporads with micronucleus while only 20% showed normal tetrads.

Fig. 5. Sporad analysis in P. ‘Fuller’s Sunset’ (a, b) and P. ‘Geumgongju’ (c, d). Most sporads formed in P. ‘Fuller Sunset’ were normal tetrads while a high frequency of micronuclei was observed in P. ‘Geumgongju’. Arrows indicate the presence of micronuclei. Scale bar=10µm.

Polyploidization plays a substantial role in the production of standard-type white Phalaenopsis cultivars (Griesbach 1985). As the demand increased to create novel Phalaenopsis cultivars with diverse colors and patterns with spots, red, yellow, and orange flowers, hybridization of species between different sections or subgenera has been implemented (Chuang et al. 2008, Tang & Chen 2007). In the 1970s, several novelty-type cultivars were produced by crossing diploid species in the subgenus Polychilos¸ including P. amboinensis, P. fasciata, P. lueddemanniana, and P. reichenbachiana (Freed 1980a, 1980b, 1981a, 1981b, Lee & Chung 2021) with standard type cultivars. These star-shaped flowers contributed to the production of yellow, spotted, and red flowers in the progeny. Polychilos normally possess asymmetrical and bimodal karyotypes (Lee et al. 2017). However, one difficulty often faced by breeders is the reduced fertility of these interspecific hybrids due to their chromosomal composition, such as triploidy and aneuploidy (Aoyama 2010), and abnormal meiotic behavior caused by the low genomic affinity between parents (Arends 1970, Bolanos-Villegas et al. 2008). Lee et al. (2017) found that Phalaenopsis triploid cultivars with small, medium, and large chromosomes were associated with low fertility. The sterility of these hybrids may be a result of the incompatibility of the parental genomes (Aalto et al. 2013). Therefore, knowledge of chromosome number, meiotic behavior, and pollen fertility is critical for determining the genetic variability of species or hybrids, and parent selection should be included in breeding programs.

In this study, hypotetraploidy and asymmetrical chromosomes were observed in P. ‘Fuller’s Sunset’ (P. ‘Taisuco Date’×P. ‘Chian Xen Queen’) and P. ‘Geumgongju’ (P. ‘Fuller’s Eldorado’×P. ‘Sin-Yaun Golden Beauty’) (RHS 2004 & 2008). P. ‘Fuller’s Sunset’ was confirmed to have a chromosome number of 2n=4x-1=75, while P. ‘Geumgongju’ had 2n=4x-2= 74. Hybrids are among the most commercially valuable orchids. They can exhibit tetraploidy, high ploidy, and even aneuploidy (Kuo et al. 2005). Aneuploid genomes have incomplete sets of chromosomes that can either occur naturally in plant populations or can be induced using physical or chemical agents (Henry et al. 2010, Pavlikova et al. 2017, Shoemaker et al. 1996). This may lead to variations in the appearance and reproductive behavior of the plant. Hybridization and endopolyploidy are the main mechanisms underlying natural polyploidization in Phalaenopsis (Vilcherrez-Atoche et al. 2022). In a study by Lee et al. (2020), 32% of the representative Phalaenopsis cultivars analyzed were found to be aneuploids, most of which were tetraploid. Most Phalaenopsis aneuploids analyzed by Kamemoto et al. (1961) had chromosomal numbers between 60 and 73. It is likely that several crosses using triploids and tetraploids produced individuals with numbers between triploid and tetraploid levels. A review of the breeding history of yellow Phalaenopsis cultivars was previously published by Lee and Chung (2021), who indicated that these cultivars carry different numbers of large chromosomes from Polychilos section species in their chromosome constitutions. This may have affected the color intensity depending on chromosome introgression during hybridization. Identification of breeding stocks that produce the desired quality of progenies and possess high fertility is of considerable importance. Mapping the chromosomes that carry the genes of interest, e.g. the genes responsible for yellow pigment expression, would be beneficial for breeding Phalaenopsis cultivars by using cytogenetic techniques to analyze individual chromosomes, chromosomal segments, or the genomes of hybrid plants (Younis et al. 2015).

Choosing genotypes with a high percentage of viable gametes is the key to effective breeding (Techio et al. 2006). To date, there has been no published data on the crossing ability of these two yellow Phalaenopsis cultivars. Nevertheless, pollen viability tests can be used to rapidly assess the viability and vigor of pollen grains. These tests are essential because they reduce the time required to create new hybrid cultivars when hybrid plants with unviable pollen grains are rejected. Therefore, only plants with more stable genotypes are selected for breeding (Lavinscky et al. 2017). In orchids, pollen tetrads develop into mature structures called compound pollen grains, which unite to form pollinia (Rao & Chin 1973).

In this study, in vitro pollen germination testing in addition to TTC staining was conducted to evaluate the pollen viability of P. ‘Fuller’s Sunset’ and P. ‘Geumgongju’. In P. ‘Fuller’s Sunset, both tests indicated the presence of viable pollinia, as confirmed by the stained pollinia in all flowering stages, as well as the growth of the pollen tubes as shown in the in vitro pollen germination. In contrast, P. ‘Geumgongju’ showed unviable pollinia in all flowering stages except during anthesis in both viability tests.

The TTC test is credible and extensively used, based on the principle that metabolically active tissues exhibit respiration and are capable of reducing a colorless chemical (TTC) into an insoluble, red-colored compound (formazan) via hydrogen transfer reactions catalyzed by dehydrogenases. Thus, it is sometimes used in staining tissues with biotic tolerance (Cho et al. 2020, El-Rawy et al. 2018). Formazan stains living tissues red. Therefore, the viable parts of the pollen should stain red when they are incubated in TTC solution (Smith 1951). Conversely, in vitro germination uses an artificial medium that simulates the conditions of style sigma to germinate the pollen and determines the germinability and growth of the pollen tubes (Rodriguez-Enriquez et al. 2013). Pollen germinates in different types of media, such as a simple sucrose/boric acid medium (Linskens 1967), similar to those used in this study, to a more complex medium containing polyethylene glycol and/or various amino acids (Read et al. 1993, Shivanna et al. 1997, Zhang & Croes 1982). Although pollen staining and in vitro pollen germination are convenient for assessing pollen viability, hand pollination is still the most effective way to check the actual potential of pollination ability (Lyakh et al. 1998).

In our cytological analysis, most tetrads in both cultivars consisted of four individual microspores. However, additional micronuclei were mostly observed in P. ‘Geumgongju’, with 80% frequency. The higher the frequency of normal pollen tetrads, the higher the probability that they contribute to the seed set (Hsu et al. 2010). Tetrads containing micronuclei may signify incomplete or unbalanced chromosomal complements and are commonly observed in Phalaenopsis hybrids. Given these unfavorable effects, pollen development may be impaired. This usually indicates poor viability and frequent capsule abortion (Bolanos-Villegas et al. 2008). In some xBrassicoraphanus lines, frequent formation of micronuclei was observed and positively correlated with pollen deformation. This implied that the presence of micronuclei induced by abnormal meiotic behavior severely affected pollen viability (Shin et al. 2021). Successful meiosis is a prerequisite for producing fertile pollen during plant reproduction. Meiotic chromosomal behavior affects pollen viability (Palma-Silva et al. 2008). Irregular meiotic pairings lead to unpaired univalents, homoeologous bivalents, or multivalents that cause missegregation of chromosomes, resulting in compromised pollen fertility (Cifuentes et al. 2010, Szadkowski et al. 2010). Analysis of the meiotic process of P. ‘Fuller’s Sunset’ and P. ‘Geumgongju’ may provide further information and clarification regarding their fertility.

Knowledge of the cytogenetics, pollen viability, and meiotic behavior of the two yellow Phalaenopsis cultivars provides valuable information for the proper planning and selection of parents. This can ensure greater success rates in achieving the specific aims of orchid hybridization. The cytogenetic background of the cultivars provided in this study is highly beneficial for planning systematic breeding programs in Phalaenopsis.

This work was supported by the Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01502405) of the Rural Development Administration of the Republic of Korea.