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Article

Trichostatin A Induced Microspore Embryogenesis and Promoted Plantlet Regeneration in Ornamental Kale (Brassica oleracea var. acephala)

by
Chuanhong Liu
1,†,
Gengxing Song
1,†,
Yonghui Zhao
1,
Bing Fang
2,
Zhiyong Liu
1,
Jie Ren
1,* and
Hui Feng
1,*
1
Department of Horticulture, Shenyang Agricultural University, No. 120 Dongling Road, Shenhe District, Shenyang 110866, China
2
Foreign Language Teaching Department, Shenyang Agricultural University, No. 120 Dongling Road, Shenhe District, Shenyang 110866, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2022, 8(9), 790; https://doi.org/10.3390/horticulturae8090790
Submission received: 20 July 2022 / Revised: 27 August 2022 / Accepted: 28 August 2022 / Published: 30 August 2022
(This article belongs to the Special Issue Innovation in Propagation and Cultivation of Ornamental Plants)
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Abstract

:
Cut flower ornamental kale (Brassica oleracea var. acephala) is a biennial cultivar, which completes a sexual reproductive generation in two years. Isolated microspore culture (IMC) can accelerate plant homozygosity instead of self-pollinations. However, the application of IMC in cut flower ornamental kale was rare since its low rate of embryogenesis. It is proved that histone acetylation might affect the gene expression in microspores and led to the transformation of microspores from pollen development pathway to embryogenesis. In this paper, microspores, derived from three varieties of cut flower ornamental kale, Crane Bicolor (CB), Crane Pink (CP) and Crane Feather Queen (CFQ), were treated with histone deacetylation inhibitor (HDACI) trichostatin A (TSA). Results revealed that the appropriate concentration of TSA was 10 nM for CB with obtaining 5.39 embryos per bud, while for CP and CFQ was 5 nM with acquiring 10.89 and 16.99 embryos per bud, respectively. TSA treatment also reduced the embryonic mortality, of which 10 nM TSA treatments CB was the optimal and the embryonic mortality decreased to 25.01%. The double haploid (DH) proportion of regenerated plants reached 37.3%. These results contribute to improving the technology for IMC in cut flower ornamental kale.

1. Introduction

Cut flower ornamental kale (Brassica oleracea var. acephala) is a 2-years herbaceous cultivar, whose leaves are colorful and changeable, with compact plant morphology and gorgeous inner leaves. Cut flower ornamental kale is mainly distributed in temperate regions, and is planted more in the United Kingdom, the Netherlands, Germany, and the United States. The ornamental period on land is over 4 months since its strong cold resistance and easy cultivation [1]. Cut flower Ornamental Kale contains a large amount of anthocyanins, which as effective antioxidants have the potential to prevent some cancers, cardiovascular diseases and other chronic diseases [2,3,4]. Cut flower ornamental kale can be self-pollinated or cross pollinated. It normally takes 5–7 years to obtain homozygous parent lineages through self-pollination from a hybrid. Isolated microspore culture (IMC) can quickly purify genotypes and accelerate breeding process [5].
IMC was first reported by Nitsch [6], and then scholars from worldwide made in-depth discussions and innovations on this technology. There are many factors that affect the IMC, involving internal factors, such as genotype, microspore viability, growth conditions, and physiological status of the material, while external factors such as donor environment, phytorregulators treatment, period of sampling and composition of the medium, etc. [7]. In addition, there are several additive substances to the culture media which can be used to improve or induce microspore embryogenesis. Tuteja et al. [8] found that surfactant treatment could promote plant tissue growth and increase cell viability. Nonionic surfactant treatment (Tween-20, Triton X-100, Pluronic F-68) can improve the microspore embryo induction rate and plant regeneration rate of purple flowering stalk (Brassica campestris ssp. chinensis var. purpurea Bailey) [9]. Chen et al. [10] discovered that 10 nM methylene blue treatment of ornamental kale microspores could increase the microspore induction rate to 17.15 embryos per bud, which was 5 times that of the control group. Physical treatments applied to the microspores or bud flowers also promoted microspore sporophytic-pathway. Ari et al. [11] reported that 4 °C cold shock treatment could promote the microspore embryogenesis rate of ornamental kale. We had tested the microspore culture of cut flower ornamental kale and detected that microspore embryogenesis was greatly difficult.
Histone deacetylation inhibitor (HDACI) is a class of compounds that change the gene expression pattern in cells by increasing the acetylation degree of histones in cells [12]. Trichostatin A (TSA) is a kind of HDACI that induces cell apoptosis, cell differentiation, regulate transcription, reversal of changed cell morphology and cell cycle arrest [13]. The function of TSA in plants was to reduce DNA methylation [14] and increase histone acetylation, resulting in changes in gene expression [15]. TSA treatment enabled recalcitrant Arabidopsis microspores to develop into embryogenic clusters. TSA treatment prevented HDAC activity in IMC of Brassica napus and induced male gametophyte to embryogenesis [16]. TSA could induce embryogenesis in wheat [12].
In this paper, the effects of different concentrations of TSA and genotypes on microspore embryo induction rate and plant regeneration in cut flower ornamental kale were investigated. The ploidy of regenerated plants was detected by flow cytometry and the horticultural characters of DH lines were investigated. This study can accelerate the breeding process and improve the breeding efficiency of cut flower ornamental kale.

2. Materials and Methods

2.1. Plant Materials

Cut flower ornamental kale F1 plant Crane Bicolor (CB), Crane Pink (CP) and Crane Feather Queen (CFQ) of Japan TAKII Company was used for IMC. CB showed bicolor round (white outside, pink inside). CP showed pink round leaves. CFQ showed red pinnate. In the autumn of 2020 (mid-July), the seeds were sown in the experimental base of the Shenyang Agricultural University. The seedlings were transplanted into 20 cm-diameter plastic pots in August (about 25 days), then plants vernalized in November and treated with 16 h long sunshine. The plants were moved to greenhouse in early December (about 140 days) in winter. From January to February (about 165–200 days) in spring, the plants bolted and bloomed, and in a sunny day, inflorescences with good growth conditions were selected for IMC.

2.2. IMC

IMC experiment was carried out after modification on the basis of Sato et al. [17] and Hoseini et al. [18]. Buds with a ratio of petal to anther length (P/A) of 0.5–0.8 were treated in a 4 °C refrigerator for 24 h. Buds were soaked in 75% (v/v) ethanol for 30 s and 0.1% (w/v) HgCl2 for 6 min in turn for disinfection, afterwards rinsed with germ-free ultrapure water for 5 min, repeated 3 times. To isolate microspores, the buds were squeezed in 8 ml B5 liquid medium with sterile glass rods. The suspensions were first filtered through a 74 μm stainless steel cell sieve and then through a 40 μm cell sieve, later collected in a 50 mL centrifuge tube and for centrifugation (2000 rpm, 3 min). The precipitates were resuspended into NLN-15 medium [19] at a cell density of 1 × 105 microspores/mL. The NLN medium containing microspores was sub-packed into sterile Petri dishes, 5 mL per dish, and 100 μL activated carbon (10 g/L) was added to each dish. The microspores were cultured at 33 °C for 1 d, finally transferred to dark culture (25 °C).

2.3. TSA Treatments

TSA was dissolved in dimethyl sulfoxide (pH 5.8). TSA concentrations were adjusted to 0, 5, 10, and 15 nM, respectively. In the process of NLN subpackaging to plastic petri dishes of IMC experiment, TSA with different concentrations was instilled.

2.4. Embryo Germination and Plantlet Regeneration

After 20 days of microspore culture, the embryos developed to approximately 0.5 cm in length, which were counted and shook (45 rpm, 25 °C) for 7 days. Whereafter, embryos were planted to solid Murashige and Skoog (MS) medium (pH 5.8, 30 g/L sucrose, 0.1 g/L AC, 7.5 g/L agar powder) [19] in conical flask. After callus formation, they were cut and moved to solid differentiation MS medium (5.5 g/L agar powder).

2.5. Ploidy Identification

The ploidy of regenerated seedlings was identified by FACSCalibur flow cytometer. The leaves were cut into 2 cm2 and put into Petri dishes containing chopping buffer solution (15 mM β-mercaptoethanol, 20 mM NaCl, 0.1% (v/v) TritonX-100, 0.5 mM spermine, 80 mM KCl, 15 mM Tris, and 2 mM EDTA-2Na at pH 7.2–7.5). Leaf samples were cut into pieces with scissors, later on, leached with a 300 mesh sieve to centrifuge tubes, centrifuged (10,000 rpm, 10 min). The supernatant was mixed with 1 mL PI dye and darkened for 15 min, after which the mixture was leached with a 500 mesh sieve. The DNA content of standard diploid was applied as control.

2.6. Experimental Design and Data Analysis

TSA treatment was repeated three times. When embryos grew to about 0.5 cm in length, the number of embryos was counted. The embryo yield measurement was to count the number of microspore embryos produced by per bud. Statistical evaluation was performed by SPSS software. To test whether the data difference was significant, Duncan’s least significant range test (p = 0.05) was used to separate the means.

3. Results

3.1. Microspore Embryogenesis of Different Genotypes

Microspore-derived embryos could be acquired from microspore culture of three genotypes (Table 1). The microspore embryogenesis rate of CB was the minimum, reaching 2.67 embryos per bud (Figure 1a), followed by CP, which was 6.20 embryos per bud (Figure 1c), the minimum was CFQ, which was 13.27 embryos per bud (Figure 1e). Significant differences were sensed in embryo induction rate among CFQ, CP and CB.

3.2. Effects of TSA on Microspore Embryogenesis

TSA advanced microspore embryogenesis in cut flower ornamental kales (Table 2). The induction rate of microspore embryos increased by 2.02-, 1.76- and 1.28-fold, compared with CB (Figure 1a), CP (Figure 1c) and CFQ (Figure 1e) controls, respectively. The concentration of the highest embryo yield was 10 nM, 5 nM and 5 nM for CB, CP and CFQ, respectively. The highest microspore embryogenesis of CB (Figure 1b) was observed at 10 nM TSA, which were 5.39 embryos per bud. 5 nM TSA peaked the number of embryos of CP (Figure 1d) and CFQ (Figure 1f), the microspore embryogenesis were 10.89 and 16.99 embryos per bud, respectively. High TSA concentration may have negative effects; for instance, the application of 10 nM TSA reduced the number of microspore embryos of CP and CFQ, which were 0.89- and 0.92-fold decreased, compared with the respective controls; in the application of higher concentration 15 nM TSA also reduced the number of microspore embryos in relation to CP and CFQ by 0.95- and 0.90-fold decreased, versus the control group.

3.3. Effects of TSA on Microspore Plant Regeneration

TSA increased direct transformation rate of microspore embryos into seedlings and reduced the mortality of microspore embryos (Table 3). To regenerate the embryos, they were transferred into MS medium (Figure 2a). Cotyledon embryos had two developmental pathways, callus formation (Figure 2b) and direct seedling formation (Figure 2c), then developed into normal plants (Figure 2d). In genotype CB, 10 nM TSA peaked the ratio of directly developed into plants at 18.22% and 4.94-fold higher versus the control group, while the proportion of embryos directly transformed into callus and embryo mortality were reduced, which were 56.77% and 25.01%, 0.91- and 0.74-fold lower, in comparison to the control group, respectively. As soon as the concentration of TSA coming up to 5 nM, the ratio of CP and CFQ directly transformed into seedlings at the highest level of 23.36% and 22.66%, respectively, which were 3.08- and 2.27-fold higher versus the control group. The direct embryo transformation rates of CP and CFQ were weaken to 47.02% and 47.61%, 0.84- and 0.91-fold lower than the control group, while the mortality was shortened to 29.32% and 30.03%, 0.81- and 0.80-fold less than the control group, respectively.

3.4. Ploidy Identification of Regenerated Plants

Fresh leaves of regenerated plants from CB, CP and CFQ were employed to recognize ploidy by FCM. Peaks appeared at the abscissa of 100, 200 and 400, representing haploid (Figure 3a), diploid (Figure 3b) and tetraploid (Figure 3c), respectively. The flower size of regenerated plants with individual ploidy was likewise diversified (Figure 3d). The highest proportion of regenerated plants was haploid, 61.0–76.6% (Table 4). Followed by DH with percentage of 21.0–37.3%, the number of tetraploid plants was the valley, range from 1.7–4.1%.

3.5. Horticultural Characteristics of DH Lines

The horticultural characters of DH lines of cut flower ornamental kale displayed heterogeneous changes. Leaf color, leaf length and plant height turning time all revealed miscellaneous degrees of variation. The DH lines of CB, CP and CFQ had compact plant morphology variation. The DH lines of CB had wavy round leaf shape, yellowish green outer leaf color, pink, white, red as well as pink inter leaf color variation (Table 5) (Figure 4). The DH lines of CP had pink, white, green, red and white inter leaf color variation (Table 6) (Figure 5). The DH lines of CFQ had grayish green and yellowish green outer leaf color, red and white, pink and white inter leaf color variation (Table 7) (Figure 6).

4. Discussion

In this paper, the microspore embryogenesis rate and plant regeneration of CB, CP and CFQ were studied by adding various concentrations of TSA to NLN medium. The results manifested that the induction rate of embryos for diversified genotypes was varying, while CFQ had the most embryos, which was 13.27 embryos per bud. 5 nM TSA treatment of CFQ yielded the most embryos with 16.99 embryos per bud.
IMC can rapidly homozygous genotyping, promoting plant embryogenesis and improving plant regeneration rate. So far, IMC has been applied to many more species [20], such as Brassica [21,22,23], barley (Hordeum vulgare L.) [24], triticale (Secale cereale) [25], eggplant (Solanum melongena L.) [26], pepper (Capsicum frutescens L.) [27], sweet pepper (Capsicum frutescens L.) [28] wheat (Hordeum vulgare L.) [29], rice (Oryza sativa L.) [30], potato (Solanum tuberosum L) [31], rye (Secale cereale L.) [32]. IMS changes the normal development direction of pollen to form haploid plants, which requires a variety of factors to regulate and control together. The genotype of the tested material affects both microspore embryogenesis and embryo yield [33]. Microspore embryo induction of 15 genotype Chinese cabbage cultivars was analyzed, of which 13 cultivars obtained microspore embryos [34]. Zeng et al. [35] found that only 7 of the 11 genotypes could obtain microspore embryos in the study of stalks. Fang et al. [36] applied 5 genotypes of flowering Chinese cabbage for IMC, and significant differences were sensed embryo induction rate among sundry genotypes. In this paper, three genotypes of cut flower ornamental kale cultivars were employed for IMC, and the embryo induction rates among the three genotypes were significantly different. Different genotypes of the same species have different ability to induce microspore embryos. This indicated that there was genotype dependence in the IMC of cut flower ornamental kale.
Microspores, as important morphologies throughout the plant life cycle, involve complex epigenetic modifications in both the normal gametophytic pathway and the stress-induced embryogenesis pathway. The impact of histone modification on embryonic development has long been concerned. Histone acetylation is regulated by histone acetylase and histone deacetylase (HDAC). Histone acetylation levels are altered when the activity of histone deacetylases is inhibited, thus affecting gene expression in microspore. It leads to the transformation for most cells from development to pollen pathway to embryogenesis, and eventually improves the embryogenesis rate of microspore [37]. TSA, a metabolite of streptomycin, is one of many HDACI at present. Li et al. [16] found that TSA treatment of Brassica napus microspore could achieve the same effect of increasing embryo induction rate as heat shock stress. Castillo et al. [38] found that 0.4 µM TSA and 0.7 m mannitol treated the anthers of bread wheat to obtain a four fold increase in the number of green DH plants. Wang et al. [39] found that 0.01 µM TSA promotes wheat embryogenesis and green plant regeneration. Zhang et al. [37] found that 0.05 µM TSA enabled pakchoi to produce the highest embryo yield and the highest plant regeneration frequency. Jiang et al. [12] found that when wheat microspores were treated with TSA, low concentration of TSA had a promoting effect, and higher dosage of TSA had no effect or harm on embryonic development, 0.1 μM TSA treatment had the most significant effect. Low concentration of TSA can promote microspore embryogenesis In this study, we treated the microspores of CB, CP and CFQ with TSA, and the results depicted that 5 nM TSA had the highest embryogenesis rate. Very low concentrations of TSA could promote ‘CB’, ‘CP’ and ‘CFQ’ embryogenesis. Chen et al. [10] also found that ornamental kale is low androgenic response: 10 nm Methylene blue can promote the embryogenesis of ornamental kale. However, high concentrations of TSA likewise inhibited microspore induction. TSA was the first application of IMC in cut flower ornamental kale.
The use of hybrids as microspore culture materials can create abundant pure and mutant materials. CB with red leaves can yield materials with pink, white and green leaves. CP with pink leaves can generate materials with green, red and white leaves. CFQ with red leaves can manufacture materials with pink, red and white leaves. Plants with green leaves can produce grayish green and yellowish green materials. Compact morphology plants can be obtained from plants with loose morphology. Plants with round leaves can produce wavy round leaf materials. The leaf color turning time is likewise advanced or delayed. Homozygous lines with abundant variation are important for breeding cut flower ornamental kale. All gene loci of DH line of cut flower ornamental kale are homozygous and can be stably inherited. It is a valuable material for selecting combinations with high yield, high quality and strong superiority.
In this research, a suitable agent for promoting embryogenesis was discovered and the appropriate concentration for cut flower ornamental kale was tested, which was of great significance for the application of IMC in cut flower ornamental kale. This study contributed to improving the technology for IMC in cut flower ornamental kale. In the next step, DH line will be used as the parent to prepare hybrid combinations, determine their combining ability and screen excellent hybrids.

5. Conclusions

This study investigated the effects of different concentrations of Trichostatin A on microspore embryogenesis and plant regeneration of cut flower ornamental kale. An effective IMC protocol for the recalcitrant genotype of cut flower ornamental kale was established. DH lines with abundant phenotypic variation were obtained. This technology can accelerate the breeding process and improve the breeding efficiency of cut flower ornamental kale.

Author Contributions

J.R., H.F. and C.L. designed the experiments. C.L., G.S., Z.L. and Y.Z. helped transplant plantlets. C.L. performed the experiments, conducted the data analysis, and wrote the manuscript. H.F. and B.F. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

The work was supported by the National Natural Science Foundation of China (Grant No. 32002070), the earmarked fund for CARS-23, and Liaoning Natural Science Foundation (2021-BS-138).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Microspore-derived embryos in three genotypes of cut flower ornamental kale treated with TSA. (a) CB with 0 nM TSA. (b) CB with 10 nM TSA. (c) CP with 0 nM TSA. (d) CP with 5 nM TSA. (e) CFQ with 0 nM TSA. (f) CFQ with 5 nM TSA. Bars = 5 cm. CB: Crane Bicolor. CP: Crane Pink. CFQ: Crane Feather Queen.
Figure 1. Microspore-derived embryos in three genotypes of cut flower ornamental kale treated with TSA. (a) CB with 0 nM TSA. (b) CB with 10 nM TSA. (c) CP with 0 nM TSA. (d) CP with 5 nM TSA. (e) CFQ with 0 nM TSA. (f) CFQ with 5 nM TSA. Bars = 5 cm. CB: Crane Bicolor. CP: Crane Pink. CFQ: Crane Feather Queen.
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Figure 2. Microspore derived plants of CFQ, CP and CB. (a) Microspore-derived embryos. (b) Callus. (c) Direct conversion to plantlet. (d) Regenerated plantlet. Bars = 1 cm. CB: Crane Bicolor. CP: Crane Pink. CFQ: Crane Feather Queen.
Figure 2. Microspore derived plants of CFQ, CP and CB. (a) Microspore-derived embryos. (b) Callus. (c) Direct conversion to plantlet. (d) Regenerated plantlet. Bars = 1 cm. CB: Crane Bicolor. CP: Crane Pink. CFQ: Crane Feather Queen.
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Figure 3. Ploidy identification of regenerated plants. (a) Haploid measured by FCM. (b) DH measured by FCM. (c) Tetraploid measured by FCM. (d) Flowers of original plant and different ploidy (original plant, haploid, Double haploid and tetraploid, from left to right). Bars = 3 mm. FCM: FACSCalibur flow cytometer.
Figure 3. Ploidy identification of regenerated plants. (a) Haploid measured by FCM. (b) DH measured by FCM. (c) Tetraploid measured by FCM. (d) Flowers of original plant and different ploidy (original plant, haploid, Double haploid and tetraploid, from left to right). Bars = 3 mm. FCM: FACSCalibur flow cytometer.
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Figure 4. DH lines of CB. (a) ‘CB-1’. (b) ‘CB-2’. (c) ‘CB-3’. (d) ‘CB-4’. (e) ‘CB-5’. (f) ‘CB-6’. Bars = 3 cm. DH: double haploid. CB: Crane Bicolor.
Figure 4. DH lines of CB. (a) ‘CB-1’. (b) ‘CB-2’. (c) ‘CB-3’. (d) ‘CB-4’. (e) ‘CB-5’. (f) ‘CB-6’. Bars = 3 cm. DH: double haploid. CB: Crane Bicolor.
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Figure 5. DH lines of CP. (a) ‘CP-1’. (b) ‘CP-2’. (c) ‘CP-3’. (d) ‘CP-4’. (e) ‘CP-5’. (f) ‘CP-6’. Bars = 3 cm. DH: double haploid. CP: Crane Pink.
Figure 5. DH lines of CP. (a) ‘CP-1’. (b) ‘CP-2’. (c) ‘CP-3’. (d) ‘CP-4’. (e) ‘CP-5’. (f) ‘CP-6’. Bars = 3 cm. DH: double haploid. CP: Crane Pink.
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Figure 6. DH lines of CFQ. (a) ‘CFQ-1’. (b) ‘CFQ-2’. (c) ‘CFQ-3’. (d) ‘CFQ-4’. (e) ‘CFQ-5’. (f) ‘CFQ-6’. Bars = 3 cm. DH: double haploid. CFQ: Crane Feather Queen.
Figure 6. DH lines of CFQ. (a) ‘CFQ-1’. (b) ‘CFQ-2’. (c) ‘CFQ-3’. (d) ‘CFQ-4’. (e) ‘CFQ-5’. (f) ‘CFQ-6’. Bars = 3 cm. DH: double haploid. CFQ: Crane Feather Queen.
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Table
Table 1. Microspore embryogenesis of CFQ, CP and CB.
Table 1. Microspore embryogenesis of CFQ, CP and CB.
GenotypeNo. of Embryos per Bud
Crane Feather Queen13.27 ± 0.28a
Crane Pink6.20 ± 0.21b
Crane Bicolor2.67 ± 0.11c
Significant differences existed in different letters (p = 0.05).
Table
Table 2. Effect of TSA concentration on microspore embryogenesis of CFQ, CP and CB.
Table 2. Effect of TSA concentration on microspore embryogenesis of CFQ, CP and CB.
HDACIConcentrationNo. of Embryos per Bud ± SD
nmol/LCrane BicolorCrane PinkCrane Feather Queen
TSA02.67 ± 0.11c 6.20 ± 0.21b13.27 ± 0.28b
53.13 ± 0.87b10.89 ± 0.35a16.99 ± 0.12a
105.39 ± 0.34a5.52 ± 0.28c12.19 ± 0.16c
152.83 ± 0.19bc5.88 ± 0.10bc11.97 ± 0.12c
Significant differences existed in different letters (p = 0.05). HDACI: histone deacetylation inhibitor. TSA: trichostatin A.
Table
Table 3. Effects of TSA concentration on plant regeneration of CFQ, CP and CB.
Table 3. Effects of TSA concentration on plant regeneration of CFQ, CP and CB.
HDACIGenotypeConcentration nmol/LRate of Direct Conversion to Plants (%)Rate of Embryos Direct Conversion to Callus (%)Rate of Embryo Death (%)
TSACrane Bicolor03.69 ± 0.09c62.66 ± 0.25a33.65 ± 0.22b
56.20 ± 0.11b62.91 ± 0.22a30.89 ± 0.65c
1018.22 ± 0.35a56.77 ± 0.44c25.01 ± 0.34d
152.66 ± 0.03d61.31 ± 0.32b36.03 ± 0.25a
Crane Pink07.69 ± 0.32b56.29 ± 0.12c36.02 ± 1.03a
523.66 ± 1.22a47.02 ± 0.55d29.32 ± 0.22c
107.22 ± 0.02b60.68 ± 1.33a32.10 ± 0.23b
155.98 ± 0.36c58.66 ± 0.19b35.36 ± 0.12a
Crane Feather Queen09.85 ± 0.33b52.53 ± 0.66c37.62 ± 0.12b
522.36 ± 1.89a47.61 ± 1.03d30.03 ± 0.11d
108.25 ± 0.35c56.49 ± 0.32a35.26 ± 0.34c
156.20 ± 0.32d54.25 ± 0.54b39.55 ± 0.36a
Significant differences existed in different letters (p = 0.05). HDACI: histone deacetylation inhibitor. TSA: trichostatin A.
Table
Table 4. Ploidy identification of microspore regenerated plants of CFQ, CP and CB.
Table 4. Ploidy identification of microspore regenerated plants of CFQ, CP and CB.
GenotypeNo. of Regeneration PlantsHaploid Double HaploidTetraploid
No. of PlantsRatio (%)No. of PlantsRatio (%)No. of PlantsRatio (%)
Crane Bicolor593661.02237.311.7
Crane Pink1249576.62621.032.4
Crane Feather Queen19613669.45226.584.1
Table
Table 5. Horticultural characters of DH line in CB. DH: double haploid. CB: Crane Bicolor.
Table 5. Horticultural characters of DH line in CB. DH: double haploid. CB: Crane Bicolor.
DH LinePlant MorphologyLeaf ShapeOuter Leaf ColorInter Leaf ColorPlant Height (cm)Leaf Length (cm)Time for Leaf Color Conversion (d)
F1looseround leafgreenred30.0120.0565
CB-1compactround leafgreenred31.2521.0265
CB-2loosewavy round leafgreenpink27.7633.6968
CB-3compactwavy round leafgreenwhite24.2821.4675
CB-4loosewavy round leafyellowish greenred and pink24.8831.3160
CB-5loosewavy round leafgreenwhite22.9623.7666
CB-6compactround leafgreenwhite12.7320.6573
Table
Table 6. Horticultural characters of DH line in CP. DH: double haploid. CP: Crane Pink.
Table 6. Horticultural characters of DH line in CP. DH: double haploid. CP: Crane Pink.
DH LinePlant MorphologyLeaf ShapeOuter Leaf ColorInter Leaf ColorPlant Height (cm)Leaf Length (cm)Time for Leaf Color Conversion (d)
F1looseround leafgreenpink31.0321.2565
CP-1looseround leafgreenpink18.0923.1268
CP-2compactround leafgreenwhite20.1424.4773
CP-3compactround leafgreengreen20.6120.0675
CP-4looseround leafgreenred and white18.8125.1064
CP-5compactround leafgreenred and white15.6122.3368
CP-6compactround leafgreenwhite26.6428.2770
Table
Table 7. Horticultural characters of DH line in CFQ. DH: double haploid. CFQ: Crane Feather Queen.
Table 7. Horticultural characters of DH line in CFQ. DH: double haploid. CFQ: Crane Feather Queen.
DH LinePlant MorphologyLeaf ShapeOuter Leaf ColorInter Leaf ColorPlant Height (cm)Leaf Length (cm)Time for Leaf Color Conversion (d)
F1loosefeather leafgreenred32.3620.3165
CFQ-1compactfeather leafgreenred and white26.3128.1065
CFQ-2loosefeather leafgrayish greenred39.2633.8166
CFQ-3loosefeather leafgreenred25.0031.2263
CFQ-4loosefeather leafgreenred and white17.9330.1168
CFQ-5loosefeather leafyellowish greenpink and white18.5932.4361
CFQ-6loosefeather leafgrayish greenred23.5142.7260
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Liu, C.; Song, G.; Zhao, Y.; Fang, B.; Liu, Z.; Ren, J.; Feng, H. Trichostatin A Induced Microspore Embryogenesis and Promoted Plantlet Regeneration in Ornamental Kale (Brassica oleracea var. acephala). Horticulturae 2022, 8, 790. https://doi.org/10.3390/horticulturae8090790

AMA Style

Liu C, Song G, Zhao Y, Fang B, Liu Z, Ren J, Feng H. Trichostatin A Induced Microspore Embryogenesis and Promoted Plantlet Regeneration in Ornamental Kale (Brassica oleracea var. acephala). Horticulturae. 2022; 8(9):790. https://doi.org/10.3390/horticulturae8090790

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Liu, Chuanhong, Gengxing Song, Yonghui Zhao, Bing Fang, Zhiyong Liu, Jie Ren, and Hui Feng. 2022. "Trichostatin A Induced Microspore Embryogenesis and Promoted Plantlet Regeneration in Ornamental Kale (Brassica oleracea var. acephala)" Horticulturae 8, no. 9: 790. https://doi.org/10.3390/horticulturae8090790

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