Cultivating Callus from Anthers and Regenerating Haploid Plants in Lilium longiflorum

Abstract

In vitro anther culture is a technique used to produce haploid plants when regenerating varieties with specific traits. To generate haploid plants with preferred characteristics, an anther culture technique was established for Lilium longiflorum “Show Up”. Morphological characteristics were recorded, including the flower bud length and anther color corresponding to different stages of microspore development. The effects of different flower bud lengths, various concentrations of exogenous plant growth regulators (PGRs), low-temperature pretreatment at 4 °C, and incubation under dark conditions on the induction of callus formation were studied. When the flower buds were 2.2–2.4 cm in length and the microspores were in the mononuclear development phase, callus induction reached the highest rate (15.6%). Callus was not induced when the PGRs 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (KT) were added separately to the growth medium, but the highest callus induction rate occurred when anthers were cultured on the medium containing 2,4-D (0.75–1.0 mg/L) and KT (4 mg/L). The low-temperature pretreatment significantly enhanced the induction rate of anthers, but prolonged low-temperature pretreatment reduced the induction rate. The optimal period of cultivation in darkness was 6 d. After 15 days of cultivation, the number of swollen anthers was recorded, and these were transferred onto the differentiation medium Murashige and Skoog (MS) + 1-naphthaleneacetic acid (NAA) (2.0 mg/L), sucrose (30 g/L), and agar (7 g/L) at pH 5.8, whereon 100% differentiation was recorded. Overall, 14 regenerated lines were obtained by in vitro anther culture. Chromosome ploidy was determined by counting chromosomes in the root tips of ten regenerated plants, and four were found to be haploids. This study lays the foundation for anther culture in lilies to shorten the breeding cycle, improve selection efficiency, facilitate efficient genetic transformation, and enable the effective production of both haploid and double-haploid plants.

1. Introduction

Lily (Lilium spp.) is an ornamental, medicinal, and edible monocot primarily distributed in Asia, Europe, and North America [1]. The genus exhibits rich diversity, with 120 known lily species worldwide. Lilies have a large genome, high heterozygosity, and complex variety ploidy [2], so excellent traits obtained through breeding can gradually be lost during cultivation [3]. The improvement of traits and selection of new varieties of lily have long relied on hybrid breeding, which is slow and costly due to the long breeding cycle and cultivation expenses [4]. Both dominant and recessive mutations can be manifested, facilitating the early identification and selection of preferred lines and allowing for diploid homozygous strains with excellent new traits to be rapidly obtained, greatly enriching plant resources [5]. Haploid plants can rapidly produce diploid homozygotes after chromosome doubling; the resulting diploid inbred lines can effectively stabilize the genetic variation carried by the haploid, thereby significantly expediting the breeding process. Haploid breeding enables the accurate selection of improved genotypes. This approach has emerged as a crucial complement to hybrid breeding methods [6]. In recent years, haploid breeding has been applied to crops such as cucumber (Cucumis sativus) [7], sweet pepper (Capsicum frutescens) [8], papaya (Vasconcellea pubescens) [9], and oat (Avena sativa) [10].

There are various ways to implement haploid breeding, among which in vitro anther culture is one of the most used methods. The earliest research on haploid lilies can be traced to 1976, when Sharp [11] cultivated haploids using the anthers of iron cannon lilies; however, their haploid traits were lost after several generations. In 1982, Gu Zhuping and Zheng Guochang [12] cultured “David” lily anthers at the late mononuclear stage by adding plant growth regulators (PGRs) to Murashige and Skoog (MS) medium to regenerate complete plants, achieving an induction rate of 8.89%; Han [13] produced haploid plants from the anthers of the Asian lily hybrid “Connecticut King” at a rate of 17.6% by adding 2.0 mg/L of picloram and 2.0 mg/L of zeatin to MS medium, and starting with anthers from another Asian lily, “Pollyanna”, as explants, Chu Yunxia [14] added 2,4-dichlorophenoxyacetic acid (2,4-D; 3.0 mg/L) and kinetin (KT; 3.0 mg/L) to the basic MS culture medium and induced haploid production at a rate of 42.16%. These findings indicate that using lily anthers for haploid culture is feasible.

Most research has focused on ovary slice culture to produce haploid lilies, and few studies have induced haploid production by in vitro anther culture, reporting low induction rates [12]. As in other genera, haploid induction in lily produces fewer varieties and currently has many limitations, such as susceptibility to the influence of germplasm type and genotype, loss of ploidy after achieving a haploid subculture, and damage to plants caused by reagent toxicity [15]. However, a newly developed breeding strategy based on haploid-induced mediated genome editing (IMGE) combines molecular and haploid breeding methods, using chromosome doubling technology to obtain double haploids (DHs) [6]. This approach not only compensates for the limitations of using existing gene-edited lines due to species and genotype dependence but also lays the foundation for the efficient and precise improvement of target genes and the commercialization of crop varieties.

In vitro anther culture has become an important supplement to traditional breeding methods, greatly improving breeding efficiency [2]. During the cultivation and doubling process, haploids may undergo genetic mutations, which can innovate germplasm resources and increase genetic diversity. Doubled haploids can accelerate the selection of pure lines, while mutated haploids enable the quick detection of recessive gene mutations, which is beneficial for mutant screening and gene function research [5].

Longiflorum hybrids of Lilium are bred from L. longiflorum and L. formosanum. The cultivars belonging to the Longiflorum hybrid exhibit the excellent characteristics of white color, elegant aroma, and superior flower type, which are suitable for cut flowers and garden cultivation, and are popular in the market at present. “Show Up” is a cultivar belonging to Longiflorum hybrids. It inherits the superior traits of L. longiflorum while exhibiting enhanced disease resistance and adaptability. Therefore, the induction of haploid plants from the “Show Up” Longiflorum hybrids holds considerable application value for breeding lily varieties that embody a serene and refined aesthetic with excellent resistance.

In this study, L. longiflorum “Show Up” anthers were used as explants to investigate the effects of flower bud length and PGRs on callus generation from anthers. The ploidy of the subsequently regenerated plants was identified to obtain complete haploid plants relatively efficiently. This study lays the foundation for shortening the breeding cycle of lilies, improving selection efficiency, obtaining homozygous offspring, and facilitating faster genetic transformation.

2. Materials and Methods

2.1. Plant Material

The lily bulbs of “Show Up” were purchased from Hongyue Flowers Limited Liability Company, Haining City, Zhajiang Province, China. The diameter of bulbs are 15–18 cm. “Show Up” plants were grown in the greenhouse at Hunan Agricultural University (28.1811° N, 113.0385° E), and flower buds of different lengths were used as the test materials. The plants were grown under controlled greenhouse conditions with a temperature range of 20–25 °C, a relative humidity of 60–70%, and a 12 h photoperiod. The soil used was a mixture of peat, perlite, and vermiculite (2:1:1), with regular fertilization and irrigation. The bulbs were planted in early March. The plants developed flower buds approximately 50 days after planting, and the buds were harvested for experimentation between 55 to 60 days post-planting. As shown in Figure 1, the flowering state of the L. longiflorum “Show Up” was displayed.

2.2. Experimental Methods

2.2.1. Observations of Lily Anther Development

Lily flower buds were classified according to length (1.8–2.0 cm, 2.0–2.2 cm, 2.2–2.4 cm, 2.4–2.6 cm, and ≥2.6 cm), and the morphological characteristics of the flower buds, anthers, and pollen were recorded. The corolla was peeled away, and the anthers were removed and fixed with Carnot fixative (anhydrous ethanol/glacial acetic acid ratio of 3:1) for 4–5 h, after which they were stored in 70% ethanol to enable microspore development to be observed [16]. During filming, the anthers were washed with distilled water, and the smear method was used to apply them to a microscope slide for observation [17]. The anthers were stained with carbolfuchsin and observed using an Olympus BX51 microscope with a DP70 camera system (manufactured by Olympus Corporation, Tokyo, Japan).

2.2.2. Initiation of Anther Cultures Containing Microspores at Different Developmental Stages

After rinsing with running water for 30 min, flower buds of different lengths (1.8–2.0 cm, 2.0–2.2 cm, 2.2–2.4 cm, 2.4–2.6 cm, and ≥2.6 cm) containing microspores at different developmental stages were disinfected on an ultra-clean workbench, placed in sterile bottles, sterilized with 75% alcohol for 30 s, soaked in 10% NaClO for 10 min, and rinsed three times with sterile water [18]. The flower buds were opened using tweezers, the stamens were removed, the filaments were discarded, and the anthers were inoculated onto the induction medium [MS + 2,4-D (1.0 mg/L) + KT (1.0 mg/L), sucrose (30 g/L), agar (7 g/L), pH 5.8]. There was no pretreatment before cultivation, and the cultivation process was carried out in a dark environment. Overall, 15 culture bottles were each inoculated with six anthers from a single flower bud. Three replicates for each flower bud length were established. Callus induction was calculated after 15 days as follows:

Induction frequency (%) = (number of induced calli/number of inoculated anthers) × 100 [13].

2.2.3. Introduction of Exogenous PGRs to Induction Medium

Based on the aforementioned experiments, the optimal stage of microspore development for callus induction and the appropriate flower bud length were identified. Buds of an appropriate length were then used as experimental materials grown in MS medium supplemented with exogenous PGRs. The effects of various concentration combinations of the PGRs 2,4-D and KT on haploid induction were compared using a two-factor, five-level orthogonal experimental design. The cultures were incubated in darkness at 25 °C. Each culture flask was inoculated with six anthers from the same flower bud, and 90 anthers were exposed to each treatment with three replicates. Callus induction was assessed after 15 days.

2.2.4. Low-Temperature Pretreatment

A low-temperature (4 °C) pretreatment was applied to anthers for durations of 0, 2, 4, 6, and 8 d. The optimal flower bud length and exogenous PGR concentrations were selected based on the results obtained from the experiments described above. The anther cultures were incubated on the MS medium containing 2,4-D (0.75 mg/L) and KT (4 mg/L) in darkness at 25 °C. Each culture flask was inoculated with six anthers from the same flower bud, and 90 anthers were exposed to each treatment with three replicates. Callus induction was assessed after 15 days.

2.2.5. Dark Culture

Anthers were incubated in darkness at 25 °C for 2, 4, 6, and 8 days or under a 12 h light/12 h dark cycle as a control condition after inoculation into the culture flasks. The optimal flower bud length, exogenous PGR concentrations, and duration of low-temperature pretreatment were selected based on the results obtained from the experiments described above. After the dark treatment, the culture bottles were transferred to light conditions with a light intensity of 1500~2000 Lx under a 12 h light/12 h dark cycle. Each culture flask was inoculated with six anthers from the same flower bud, and 90 anthers were exposed to each treatment with three replicates. Callus induction was assessed after 15 days.

2.2.6. Formation of Anther Callus Tissue and Plant Regeneration

After 15 days of culture, the swollen anthers were transferred into differentiation medium [MS + 1-Naphthaleneacetic acid (NAA) (2.0 mg/L), sucrose (30 g/L), agar (7 g/L), pH 5.8] to induce further development. The differentiation efficiency was assessed based on the number of anthers exhibiting morphological changes indicative of successful differentiation.

2.2.7. Identifying the Ploidy of Regenerated Plants

Fresh 3–4 mm root tips were cut and immediately immersed in a saturated solution of p-dichlorobenzene for 5–6 h. They were rinsed 2–3 times with distilled water, fixed with Carnoy’s fixative for 12 h, and rinsed three times with distilled water. The root tips were then isolated in 6 mol/L HCl for 6–8 min until soft. They were then rinsed three times with distilled water, stained with carbolfuchsin stain solution for 20–30 min, pressed, and examined under a microscope, and photos were taken (Olympus microscope BX51, manufactured by Olympus Corporation, Tokyo, Japan) to detect chromosomes [19].

2.2.8. Statistical Analysis

Statistical analyses were conducted using Microsoft Excel 2003 and IBM SPSS Statistics 25 software. To evaluate the significance of the experimental results, a one-way analysis of variance (ANOVA) was performed, followed by Duncan’s honestly significant difference (HSD) test for multiple comparisons of means. The ANOVA was used to determine whether there were statistically significant differences among the treatment groups, while Duncan’s HSD test was applied to identify specific differences between individual treatments. The significance level was set at p < 0.05 for all analyses. Additionally, the standard deviation (SD) and standard error (SE) were calculated to assess the variability and precision of the data. These statistical methods were chosen to ensure a robust and reliable interpretation of the experimental outcomes, particularly in evaluating the effects of different treatments on callus induction and differentiation efficiency.

3. Results and Discussion

3.1. Results

3.1.1. The Relationship Between Flower Bud Length, Anther and Pollen Color, and Microspore Development Stage

The process of meiosis is synchronous with flower bud length and pollen and anther color. As shown in

Table 1. The relationship between the length of the flower bud, the color of anther and pollen, and the stage of microspore development in L. longiflorum “Show Up”.

Length of Flower Bud/cm	Stage of Microspores Development	Anther Color	Pollen Color
1.8–2.0	Pollen mother cell	Green	White, non-transparent
2.0–2.2	Dyad, tetrad	Green	White, non-transparent
2.2–2.4	Microspore	Yellow-green	Light yellow
2.4–2.6	Two nuclear periods	Yellow-green	Light yellow
≥2.6	Pollen	Light yellow	Yellow

and Figure 2, when the length of the flower bud was 2.2–2.4 cm, microspore development was at the microspore stage, the anthers were yellow-green, and the pollen was light yellow. This synchronization suggests that the flower bud length and color can serve as reliable external indicators for determining the developmental stage of microspores, which is critical for optimizing the timing of anther culture and callus induction.

3.1.2. The Correlation Between the Different Developmental Stages of Microspores and Callus Formation

Microspores at different developmental stages were inoculated onto induction medium containing 1 mg/L 2,4-D and 1 mg/L KT. After 15 days of cultivation, the flower bud length was found to be closely related to anther swelling in vitro. As shown in

Table 2. Effects of microspore development stages on callus induction.

Length of Flower Bud/cm	Number of Inoculated Anthers	Number of Induced Calli	Induction Frequency (%)
1.8–2.0	90	0	0
2.0–2.2	90	0	0
2.2–2.4	90	14	15.6
2.4–2.6	90	0	0
≥2.6	90	0	0

, the callus was obtained only when the bud length was 2.2–2.4 cm, and the induction frequency was calculated to be 15.6%.

3.1.3. Effects of Exogenous PGRs in Varying Concentrations on Callus Formation

Table 3. Rate of anther callus induction under different concentrations of exogenous PGRs in L. longiflorum “Show Up”.

Group	Number of Inoculated Anthers	2,4-D (mg/L)	KT (mg/L)	Induction Frequency (%)
Test 1	90	0	0	0
Test 2	90	0	2	0
Test 3	90	0	4	0
Test 4	90	0	6	0
Test 5	90	0	8	0
Test 6	90	0.25	0	0
Test 7	90	0.25	2	11.10 ± 1.50 h
Test 8	90	0.25	4	17.43 ± 2.85 def
Test 9	90	0.25	6	14.80 ± 1.13 g
Test 10	90	0.25	8	8.53 ± 1.56 i
Test 11	90	0.5	0	0
Test 12	90	0.5	2	18.17 ± 1.66 cde
Test 13	90	0.5	4	19.27 ± 3.65 bcd
Test 14	90	0.5	6	16.30 ± 1.89 efg
Test 15	90	0.5	8	15.57 ± 2.25 fg
Test 16	90	0.75	0	0
Test 17	90	0.75	2	20.37 ± 1.68 bc
Test 18	90	0.75	4	25.20 ± 2.59 a
Test 19	90	0.75	6	20.73 ± 0.64 b
Test 20	90	0.75	8	17.07 ± 2.54 defg
Test 21	90	1	0	0
Test 22	90	1	2	16.33 ± 0.64 efg
Test 23	90	1	4	21.47 ± 2.21 b
Test 24	90	1	6	17.43 ± 5.45 def
Test 25	90	1	8	14.80 ± 2.77 g

Means with different letters in the same column are significantly different (Duncan LSD, p ≤ 0.05).

shows that the induction rate of the anther callus tissue varied when 2,4-D and KT were added at different concentrations. When 2,4-D or KT was added separately to the culture medium, the induction rate of callus tissue was 0%, indicating that only the simultaneous addition of 2,4-D and KT can induce anther callus tissue. The callus appears as a soft, friable, and irregularly shaped mass with a color ranging from pale yellow to light green depending on the developmental stage and culture conditions. The texture of the callus is often granular or nodular with a moist surface due to the secretion of extracellular substances. Under optimal conditions, callus formation initiates within 7 to 10 days after anther inoculation, with visible proliferation observed by 15 days. The combination of 2,4-D at 0.75 mg/L and KT at 4 mg/L led to the highest induction rate, 25.2%. ANOVA analysis and significance analysis are shown in Table S1.

3.1.4. The Effect of 4 °C Pretreatment on the Induction of Anther Callus Tissue

As shown in Figure 3, compared with no pretreatment (0 d at 4 °C), pretreatment at 4 °C improved the callus induction rate, but prolonged low-temperature pretreatment reduced the induction rate. Anthers subjected to the 4 °C pretreatment exhibited visible swelling, which correlated with higher callus formation. As shown in Figure 4, anthers pretreated at low temperature for 4 d showed better swelling effects than those pretreated for 0 d. ANOVA analysis and significance analysis are shown in Figure S1.

3.1.5. The Effect of Cultivating Anthers in Darkness on the Induction of Callus Tissue

Culturing anthers under dark conditions significantly impacted the induction of anther callus tissue (Figure 5). Without a dark period, callus tissue formation was not induced. The most suitable period of darkness for inducing callus tissue was 6 d. As shown in Figure 6, compared with an anther with dark treatment, the anther incubated without dark treatment swelled, turned green, and finally turned brown and necrotic. ANOVA analysis and significance analysis are shown in Figure S2.

3.1.6. Formation of Callus Tissue from Lily Anthers and Plant Regeneration

After 10 days of cultivation, the anthers exhibited noticeable swelling, indicating the initial stages of callus induction. After 15 days, the anthers split open, and yellow callus tissue emerged at the ventral suture, demonstrating successful callus proliferation. The anthers were transferred onto differentiation medium (MS + NAA 2.0 mg/L, sucrose 30 g/L). In the 20 days after being transferred onto differentiation medium, from the callus tissue, adventitious buds differentiated, whereon the differentiation rate reached 100%. After 30 days, the callus in the anthers was significantly enhanced, accompanied by the initiation of adventitious root differentiation. After 40 days, the callus tissue, which had developed adventitious roots and buds, was meticulously dissected from the anthers and transferred to a new culture medium. After 14 days, leaf differentiation was evident, and the root system exhibited enhanced vigor and was subsequently cultured to eventually develop into regenerated plants. As shown in Figure 7, haploid plants exhibited smaller leaves and less developed roots than normal diploid plants.

3.1.7. Plant Ploidy Identification

Chromosome detection was performed on the root tips of regenerated plants using the pressing method. Among the regenerated plants, 40% were haploids, containing 12 chromosomes (Figure 8), while diploid plants accounted for 60% and contained 24 chromosomes.

4. Discussion

Some excellent traits may appear during the natural growth and reproduction processes in lily, but most of the genes for these traits are heterozygous. Therefore, if these traits are not retained, they disappear over time through natural reproduction. This indicates that preserving excellent traits through haploid culture is becoming increasingly important to improving lilies.

In recent years, the rapid advancement of anther culture techniques has enabled their application not only for harnessing haploid individuals alongside other superior varieties to enhance plant resource collections but also for inducing chemical or physical mutations in anthers or their callus tissues. Thus, highly resistant mutants were obtained; for example, He [20] produced mutants with an oil acid content reaching 80.34% after applying ethyl methylsulfone (EMS) to induce mutagenesis in Brassica napus microspores. For breeding purposes, anther culture is frequently integrated with transgenic technologies, utilizing haploid protoplasts or embryo-like tissues generated through anther culture as intermediate substrates and employing methods such as gene gun and soaking techniques to facilitate gene transformation, ultimately resulting in the cultivation of transgenic plants.

The flower bud length corresponding to a particular microspore development stage varies among varieties of lilies. For instance, the length of the flower buds in the Asiatic lily hybrid variety “Tresor” during the meiotic stage is in the range of 1.9–2.4 cm, whereas those of the Oriental lily hybrid varieties “Sorbonne” and “Siberia” are in the ranges of 2.1–3.1 cm and 2.9–3.9 cm, respectively, and those of the LA and OT series lilies “Ceb Dazzle” and “Robina” are in the ranges of 2.0–2.8 cm and 3.0–4.0 cm, respectively [21]. Our findings indicated that the most appropriate flower bud length for anther culture in vitro was 2.2–2.4 cm in L. longiflorum “Show Up”; at this length range, the microspores were at the mononuclear microspore stage of development. Therefore, when conducting anther culture in different varieties, it is necessary to study the microspore development stage.

The selection of flower buds or anthers at the appropriate microspore development stage is a crucial factor influencing anther culture. It has been confirmed in many higher plant species that there is a distinct correlation between the microspore development stage and flower morphology. The effect of callus formation varies among microspores at different stages on the same culture medium. Among them, anthers in the middle and late stages of mononuclear growth are highly sensitive to external environmental stimuli and are the best tissue culture materials for many plants (e.g., in the in vitro culture of anthers from peppers and other plants, it is generally believed that anthers in the early mononuclear stage are the best material for selection) [22]. The optimal period for inducing embryoid development in oats is from the late mononuclear stage to the early stage of binucleate microspore development [23]. For L. longiflorum “Show Up”, the optimal period was found to be the mononuclear microspore stage, during which the flower bud was 2.2–2.4 cm in length, the outer parts of the anthers were yellow-green, and the anthers were light yellow. This experimental study revealed that during the late mononuclear stage, the microspore wall gradually thickens, which is not conducive to nutrient absorption. Therefore, choosing microspores at the early and middle mononuclear stages is more suitable for cultivation.

When culturing anthers, the concentrations and combinations of exogenous PGRs are crucial to success. Previous reports have noted that the optimal concentrations of exogenous PGRs for anther callus induction are Synthetic Nutrient Growth Medium (SNGM) + NAA (2.5 mg/L) + 6-Benzylaminopurine (6-BA) (0.5 mg/L) in Loropetalum chinense var. rubrum [24] and thidiazuron (TDZ) (0.5 mg/L) + 2,4-D (1.0 mg/L) in Dongzao (Ziziphus jujuba Mill.) [25]. Moreover, the optimal ratios of PGRs for inducing anther callus tissue formation in Lilium × formolongi “Raizan 1” are 2,4-D (0.5 mg/L) and KT (4.0 mg/L) [26]; however, the present study revealed the optimal PGR ratios to be 2,4-D (0.75 mg/L) and KT (4.0 mg/L) in L. longiflorum “Show Up”.

Most researchers agree that the pretreatment of anthers at 4 °C can significantly enhance the induction of callus formation because the low-temperature pretreatment helps to prevent the formation of spindle-shaped filaments. Wang [27] found that Actinidia arguta anthers subjected to low-temperature pretreatment for 5 d had a significantly higher callus induction rate compared with anthers pretreated for 0, 1, 3, and 7 d, and in Rhodomyrtus tomentosa anthers pretreated at 4 °C for 24 h, the rate of callus induction was significantly higher than that of anthers pretreated for 0, 12, 48, and 72 h [28]. However, low-temperature pretreatment does not always significantly improve callus induction. Xu [29] found that among the three Jujube (Ziziphus jujuba Mill.) varieties, the low-temperature treatment of anthers increased the rate of callus induction in two of them, “Dongzao” and “Qiyuexian”, but no such effect was observed in the third, “Junzao”. In this study, the callus induction rate in L. longiflorum “Show Up” after pretreating the anthers at 4 °C for 4 d was higher than those of the control and the pretreating at 4 °C for 2, 6, and 8 d.

It is generally believed that dark conditions promote callus induction in anthers, with differentiation being facilitated by a gradual increase in light intensity following cultivation in darkness. However, light is not an essential factor for the development and differentiation of callus; it primarily affects its quality. Gou [30] reported that callus induced under complete darkness appeared light yellow, granular, and shiny, while the quality of callus grown under continuous light was poor with severe browning. You [31] found that blueberry (Vaccinium) anthers produce callus under 16 h light/8 h dark culture conditions. Blueberry anthers can also form callus under dark culture conditions, with longer dark culture periods leading to easier callus induction. In this study, dark treatment for 6 d increased the rate of callus induction in L. longiflorum “Show Up”, but over longer periods, cultivation in darkness reduced the induction rate.

During cultivation to induce callus formation, the anther wall is somatic and therefore diploid or polyploid. Some of the induced callus is derived from the somatic cells in the anther wall. The plants formed from this section of the anther are not haploids, so it is necessary to identify the ploidy of all plants formed. This process is laborious and time-consuming, and the number of produced haploids is low. A known anther in vitro culture technology, isolated microspore culture, directly isolates fresh microspores from anthers or flower buds for culture without any form of preculture. The technique overcomes many of the challenges associated with anther culture as immature pollen from higher plants can be grown into haploid plants in vitro. However, isolated microspore culture has not been widely applied to flower bulbs, and there are few reports of its use in lilies [21].

Anther culture differs among lily varieties. In this study, only L. longiflorum “Show Up” anthers were used in our investigation of anther culture. Future studies should expand the scope of varieties included and explore genotypes that allow for easier induction and differentiation of callus to assist future breeding strategies for creating lily cultivars with excellent traits.

5. Conclusions

The results revealed that the mononuclear stage was the suitable microspore development stage for including anther callus formation; the optimal medium for anther callus induction was MS + 2,4-D 0.75 mg/L + KT 4.0 mg/L + sucrose 30 g/L + agar 7 g/L; low-temperature pretreatment at 4 °C could significantly improve the induction rate of anther callus formation; and the most suitable dark culture time was 6 d. Based on these findings, an efficient in vitro anther culture system for L. longiflorum “Show Up” was successfully established. In this system, the highest rate of anther callus induction of “Show Up” reached 25.2%, and the regeneration frequency of haploid plants was 40%. The successful implementation of anther culture enabled the efficient production of haploid and homozygous diploid plants. Therefore, this study lays a solid foundation for future research on lily breeding, genetic transformation, and genetic map construction.