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Efficient doubled haploid production in microspore culture of Zengcheng flowering Chinese cabbage (Brassica campestris L. ssp. chinensis [L.] Makino var. utilis Tsen et Lee)
Scientia Horticulturae 245 (2019) 57–64
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Efficient doubled haploid production in microspore culture of Zengcheng flowering Chinese cabbage (Brassica campestris L. ssp. chinensis [L.] Makino var. utilis Tsen et Lee) Liujing Niu1, Fengyan Shi1, Hui Feng, Yun Zhang
T
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Laboratory of Vegetable Genetics Breeding and Biotechnology, Department of Horticulture, Shenyang Agricultural University, No. 120 Dongling Road, Shenhe District, Shenyang, 110866, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Zengcheng flowering Chinese cabbage Microspore culture L-ascorbic acid sodium salt Embryogenesis Doubled haploid lines
Microspore culture has been implemented in breeding programs to produce doubled haploids (DH) and thus accelerate the breeding process. This study aimed to establish an efficient microspore culture protocol for Zengcheng flowering Chinese cabbage, a highly nutritious and valued vegetable, that would be suitable for heterosis breeding. Microspores of three genotypes, 17AY010, 17AY011, and 17AY012 were successfully induced to produce embryos in NLN-13 medium. Two genotypes, 17AY011 and 17AY012 were cultivated in NLN13 medium supplemented with different concentrations (0, 0.2, 1, 5 or 25 μM) of L-ascorbic acid sodium salt (Vitamin C Sodium Salt, VcNa) to enhance microspore embryogenesis and plant regeneration without an intervening callus phase. In both genotypes, the addition of 1 μM and 0.2 μM VcNa significantly increased the frequency of embryogenesis, 6.55-fold and 10.33-fold as compared with the control, respectively. The optimum concentration of VcNa application that would improve the rates of direct plant regeneration in the two tested genotypes was 1 μM. Moreover, the doubled haploid rates of regenerated plants of the three genotypes were above 60%. Five DH lines of 17AY012 with high yield were obtained. According to the measurement of the affinity index, they were prospective self-incompatible lines for hybrid breeding.
1. Introduction Zengcheng flowering Chinese cabbage (Brassica campestris L. ssp. chinensis [L.] Makino var. utilis Tsen et Lee) is a variety of Brassica vegetable crop with edible flower stalk typical for Zengcheng City in Guangdong Province, China. It is appreciated for its fresh, crisp, sweet flavor, and high nutritional value (Liu et al., 2009). As more people are attracted to this variety, its market demand and cultivation area continue to increase steadily. Zengcheng flowering Chinese cabbage is a typical cross-pollinated plant with significant levels of heterosis, and therefore homozygous parental materials play an important role in the breeding of this hybrid. Compared with conventional breeding methods, microspore culture is an effective technology for rapid development of doubled haploid lines, which can significantly accelerate the breeding process (Zhou et al., 2002; Ferrie and Caswell, 2011). Since Lichter (1982) successfully created embryos of Brassica napus L. by using microspore culture, this technique has been used in many plant species. For example, Cao
et al. (1992) produced 200 Chinese cabbage regenerated plants by isolated microspore culture, and the method has been successfully implemented in many other Brassica crops such as broccoli (B. oleracea L. var. italica, Takahata and Keller, 1991; Duijs et al., 1992), tronchuda cabbage (B. oleracea L. var. costata, Vicente and Dias, 1996), ornamental kale (B. oleracea L. var. acephala, Zhang et al., 2008), and sauerkraut cabbage (B. oleracea L. var. capitata, Cao et al., 1990). The induction frequency of microspore-derived embryos is influenced by many factors including donor plant genotype, the developmental stage of microspores, culture conditions, and culture medium (Babbar et al., 2004; Bhowmik et al., 2011; Ferrie and Caswell, 2011; Winarto and Teixeira da Silva, 2011). Many studies have shown that the addition of appropriate amount of chemical reagents to the medium can increase microspore embryogenesis. For instance, adding 1 μM of aminoethoxyvinylglycine to the NLN-13 medium can significantly increase embryo yield of Brussels sprouts (Zeng et al., 2015a), whereas addition of trichostatin A (0.05 μM), suberoylanilide hydroxamic acid (0.05 μM and 0.10 μM), and sodium butyrate (2 μM) effectively increased the
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Corresponding author. E-mail address: [email protected] (Y. Zhang). 1 Liujing Niu and Fengyan Shi contributed equally to this work and share first authorship. https://doi.org/10.1016/j.scienta.2018.09.076 Received 7 June 2018; Received in revised form 26 September 2018; Accepted 27 September 2018 0304-4238/ © 2018 Elsevier B.V. All rights reserved.
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frequency of microspore embryogenesis and plant regeneration in pakchoi (Zhang et al., 2016). In addition, Zeng et al. (2017) reported that the addition of 10 mg/L ascorbate and 20 mg/L glutathione in ½ NLN medium increased the rate of embryo induction of broccoli. Adding organelle antioxidants to the microspore induction medium of Brassica napus and white cabbage allows mitochondria to maintain normal content of reactive oxygen species (ROS), which ensures normal development of microspores, and increases the frequency of microspore-derived embryogenesis (Hoseini et al., 2014; Zeng et al., 2015b). To date, the application of microspore culture in Zengcheng flowering Chinese cabbage has not been reported. Given its limited success in Brassica rapa L., we investigated the effects of different genotypes and addition of organelle antioxidant VcNa on microspore embryogenesis and plant regeneration. The ploidy level of regenerated plants, the morphological characters and plot yield of DH lines were identified. The main purpose of this study is to establish an efficient microspore culture protocol in Zengcheng flowering Chinese cabbage and get DH lines that can be applied to the practice of heterosis breeding.
Table 2 Effect of three genotypes on microspore embryogenesis of Zengcheng flowering Chinese cabbage. Code
No. of embryos per bud ± SD
17AY010 17AY011 17AY012
0.16 ± 0.04b 0.42 ± 0.02a 0.21 ± 0.02b
Note: Means followed by different lowercase letters are significantly different at P = 0.05 level.
supernatant was discarded and the pellet was resuspended in NLN-13 medium (pH 5.84) at a cell density of 1 × 105–2 × 105 microspores·mL−1. The microspore suspension was divided into sterile plastic culture dishes (60 mm × 15 mm), 5 mL per dish, and 100 μL activated carbon solution (0.05 g agarose and 1 g activated carbon suspended in 100 mL double-distilled water) were added to each dish. Finally, the Petri dishes were sealed with Parafilm membranes and incubated at 33 ℃ for 24 h, and then transferred to an incubator, cultured at 25 ℃ in the dark. Once the embryoids were visualized to the naked eye (about 0.5 cm long, about 10 days after microspore culture), the Petri dishes were transferred to a rotary shaker (25 ℃, 50 rpm).
2. Material and methods 2.1. Plant material
2.3. Effect of genotypes on microspore embryogenesis
The heterozygous, self-incompatible donor plants were three genotypes of Zengcheng flowering Chinese cabbage that were imported from Guangdong Province, China. In order to guarantee the uniformity of the microspores for genotype and VcNa treatment, buds from all plants were selected and mixed into groups equally. The codes and basic characteristics of the three genotypes are shown in Table 1. The seeds were sown in the greenhouse at the genetic breeding experimental base of Shenyang Agricultural University in Liaoning Province. When the plants developed four leaves, about 12 cm height, they were transplanted into flowerpots with a diameter of 20 cm and cultivated in the greenhouse under a photoperiod of 16 h and at a day/night temperature of 25/15 °C. Once first flowers developed, the top inflorescences were removed, and after the lateral branches blossomed again, the buds were harvested for microspore culture.
After 21 days, the number of embryos of the three genotypes 17AY010, 17AY011, and 17AY012 was recorded and the difference in microspore embryogenesis among the genotypes was analyzed. 2.4. VcNa treatments Two varieties, 17AY011 and 17AY012, were used for VcNa treatments. The VcNa stock solution with a concentration of 200 μM was dissolved in double-distilled water. The stock solution was filter-sterilized. After dispensing 5 mL microspore suspension into the Petri dish, different volumes of VcNa were added. The final concentration in the NLN-13 medium was 0, 0.2, 1, 5 or 25 μM.
2.2. Microspore culture
2.5. Plant regeneration and ploidy level of the regenerated plants
This experiment followed the microspore culture methods described by Sato et al. (1989) and Hoseini et al. (2014). The pest- and diseasefree inflorescences were harvested on sunny days at 10:00 and pretreated at 4 ℃ for 24 h. Flower buds with the ratio of petal length to anther length of 1/3–4/5 were selected for microspore culture. The buds were sterilized in 75% ethanol (30 s), and 0.1% mercuric chloride solution (6 min) and rinsed with sterilized distilled water three times for 5 min each. The buds were macerated in 10–15 mL B5 liquid medium with 13% (m/v) sucrose, pH 5.84 (Gamborg et al., 1968) using sterile glass rods to release the microspores. The microspores were filtered into a 100 mL beaker through a 74 μm stainless steel cell sieve. The obtained microspore suspension was filtered into 50 mL centrifuge tubes through a 40 μm cell sieve and centrifuged at 2000 rpm for 3 min. The supernatant was removed and 30 mL of B5 medium was added to the pellet and the suspension was centrifuged for additional 3 min. The
The cotyledonary embryos with a length of 3–4 mm from different VcNa concentrations in the NLN medium were inoculated into the conventional solid Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) containing 3% (m/v) sucrose, 0.55% (m/v) agar, and 0.1% (m/v) activated carbon with pH adjusted to 5.84. After 3 weeks of incubation at 25 ± 1 ℃ and 16 h/8 h photoperiod, the rates of embryos directly converting to plants, formation of callus, and mortality were recorded. Flow cytometry was used to identify ploidy of plants regenerated by microspore culture for each genotype (Niu et al., 2015). 2.6. Morphological traits identification and plot yield analysis of DH lines Eleven DH lines of 17AY012 were sown in 32-hole seedling trays on 1 st August 2017, in the plastic tunnel, and after a month they were planted in the field. Complete random design was used with three
Table 1 Donor plants used for microspore embryogenesis of Brassica campestris ssp. Chinensis var. utilis. Genotype code
Variety
Characters
17AY010
sweet Zengcheng flowering Chinese cabbage
17AY011
late-maturing Zengcheng flowering Chinese cabbage
17AY012
late-maturing Zengcheng flowering Chinese cabbage, extrasweet
medium-maturing, fewer lateral tillers, bright green leaf; main stalk height 18–26 cm, main stalk width 2–4 cm late-maturing, more lateral tillers, light green leaf; main stalk height 20–25 cm, main stalk width 2–4 cm late-maturing, more lateral tillers, light green leaf; main stalk height 20–28 cm, main stalk width 2–3 cm
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Fig. 1. Embryo yield of Zengcheng flowering Chinese cabbage genotypes cultivated in NLN-13 medium supplemented with different concentrations of VcNa and embryos development. A, Embryos in the control medium (no VcNa added, 17AY011). B, Embryos in 1 μM VcNa treatment (17AY011). C, Embryos in the control medium (no VcNa added, 17AY012). D, Embryos in 0.2 μM VcNa treatment (17AY012). E, Cotyledonary embryos transferred to MS solid medium. F, regenerated plantlet.
2.7. Self-compatibility test of DH lines
replications. Fifteen plants were planted in each plot with spacing of 30 cm × 60 cm. Leaf hair, leaf color, leaf edge, leaf shape and maturity period were identified. Height and weight of the main stalk, length and width of leaf and petiole on lateral stalk were measured. After the main stalk firstly harvest, lateral stalks were collected every 5 days and collected a total of 3 times. Finally, the total yield of DH plants was calculated.
The DH lines of 17AY012 with high yield were selected to determine the self-compatibility index. After flowering, the plants were bagged 2–3 days before pollination and pollinated artificially at the bud and flowering stage. After 8–10 days, the bags were removed, and the seeds were harvested after ripening. Affinity index was used to select selfincompatible lines, and it was calculated as follows:
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10.33-fold increase when compared with the control) (Fig. 1C and D). In treatments with 25 μM VcNa, the embryo formation rates were inhibited in the two genotypes, but did not reach the significant level (Table 3). The results showed that VcNa concentration of 0.2 μM – 5 μM significantly promoted the microspore embryogenesis in Zengcheng flowering Chinese cabbage, but the optimum concentration differed between the two genotypes. The positive effects of VcNa on plant regeneration in NLN-13 medium were observed in genotypes 17AY011 and 17AY012 (Table 4). The embryos with cotyledons were transferred to MS solid medium (Fig. 1E), and regenerated plants (Fig. 1F) were formed after the embryo development. At 0.2 μM VcNa concentration and compared with the control, 17AY012 increased significantly in terms of the rate of direct conversion to plants (51.02%). In treatments with 1 μM VcNa, 17AY011 had the highest rate of direct conversion to plants (67.67%, 1.33-fold higher compared with the control) and the lowest rate of callus formation (16.24%); a remarkable increase in the rate of direct conversion to plants (52.21%, 1.33-fold higher compared with the control) was observed in 17AY012, whereas the rate of callogenesis decreased to 25.33% in 17AY012. As VcNa concentration increased to 5 μM, the rate of direct conversion to plants was not improved significantly; callus formation were significantly decreased to 23.65% in 17AY011; in 17AY012, the rate of direct conversion to plants increased to 49.00%, which was significantly different compared to the control, callus formation rate decreased to 20.07%. There was no significant difference among 0.2, 1 and 5 μM at the rates of direct conversion to plants and callus formation in 17AY012. Finally, at the highest tested VcNa concentration of 25 μM, the rate of direct conversion to plants in both genotypes was decreased, but there was no significant difference compared to the control; the mortality was increased significantly in both genotypes. To sum up, the optimal concentration of VcNa in the NLN-13 medium that would increase the rate of direct conversion to plants and reduce the formation of callus in the two genotypes was 1 μM.
Table 3 Effect of four concentrations of L-ascorbic acid sodium salt (VcNA) on microspore embryogenesis of two genotypes of Zengcheng flowering Chinese cabbage. VcNa (μM)
No. of embryos per bud ± SD
0 0.2 1 5 25
17AY011
17AY012
0.42 1.67 2.75 1.29 0.29
0.21 2.17 0.83 1.54 0.17
± ± ± ± ±
0.08c 0.17b 0.18a 0.15b 0.08c
± ± ± ± ±
0.08d 0.17a 0.11c 0.15b 0.05d
Note: Means followed by different lowercase letters in the column are significantly different at P = 0.05 level.
Affinity index = number of self-pollinated seeds/number of pollinated flowers (buds). 2.8. Experimental design and data analysis The microspore embryogenesis experiments were conducted using complete randomized design with three replicates (each replicate comprised six Petri dishes, each Petri dish contained 5 buds). Data were analyzed using SPSS (16.0) and the means were compared by using Duncan’s multiple range test at P = 0.05. 3. Results 3.1. Effect of genotypes on microspore embryogenesis in Zengcheng flowering Chinese cabbage Embryos of all three genotypes were successfully induced from microspore cultures using NLN-13 medium, but the frequency of microspore embryogenesis was low. The highest embryo induction rate was observed in 17AY011 (0.42 embryos per bud) and the lowest in 17AY010 (0.16 embryos per bud) (Table 2). There was a significant difference in the frequency of microspore embryogenesis between 17AY011 and the other two different genotypes.
3.3. Ploidy identification of regenerated plants in Zengcheng flowering Chinese cabbage The ploidy of regenerated plants was identified by flow cytometry. The histogram peaks near 100 (Fig. 2A), 200 (Fig. 2B), and 400 (Fig. 2C) indicated that the plant was haploid, doubled haploid, and tetraploid, respectively. The rates of doubling frequency of the three genotypes were more than 60%, and the highest DH rate was 71.43% (17AY010) (Table 5).
3.2. Effect of VcNa on microspore embryogenesis and plant regeneration We selected 17AY011 and 17AY012 to examine the effects of different concentrations of VcNa on the frequency of microspore embryogenesis. In 17AY011, when the VcNa at the concentration of 0.2 μM and 5 μM, the embryo induction rate increased to 1.67 and 1.29 embryos per bud, respectively. The highest frequency of microspore embryogenesis, 2.75 embryos per bud, was observed in NLN-13 medium with 1 μM VcNa; this was 6.55-fold higher than that of the control (Fig. 1A and B). In genotype 17AY012, the highest frequency of embryogenesis was obtained at 0.2 μM VcNa (2.17 embryos per bud, a
3.4. Identification of DH lines 3.4.1. Morphological traits identification of DH lines The plant morphological traits of the DH lines from 17AY012 such as presence or absence of hairs, leaf traits, maturing period, and
Table 4 The effect of VcNa on plant regeneration of two genotypes of Zengcheng flowering Chinese cabbage. Genotype
17AY011
17AY012
Concentration (μM)
Rate of embryos directly converting to plants (%)
Rate of embryos converting to callus (%)
Rate of embryos death (%)
0 0.2 1 5 25 0 0.2 1 5 25
51.00 47.67 67.67 55.34 41.67 30.67 51.02 52.21 49.00 22.33
33.23 37.33 16.24 23.65 21.00 44.30 22.35 25.33 20.07 35.62
15.77 15.00 16.09 21.01 37.33 25.03 26.63 22.46 30.93 42.05
± ± ± ± ± ± ± ± ± ±
8.23b 1.47b 3.11a 5.38ab 6.34b 3.16b 1.74a 5.37a 4.78a 4.72b
± ± ± ± ± ± ± ± ± ±
4.63a 2.66a 2.28b 4.57b 2.04b 3.62a 2.37c 3.36c 3.53c 1.48b
Note: Means followed by different lowercase letters within the genotype in the column are significantly different at P = 0.05 level. 60
± ± ± ± ± ± ± ± ± ±
6.26b 1.37b 2.25b 3.16b 1.72a 4.21b 1.07b 2.66b 5.73ab 3.42a
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Fig. 2. DNA content distribution in leaves of regenerated plants. A, Haploid. B, Doubled haploid. C, Polyploid.
CX014, CX016 and CX017 needed 97 days to be harvest that was the same with the control, the DH lines CX011, CX012 and CX018 were 110 days- maturity which was the latest. The highest main stalk was 24.46 cm (CX019) that was 3.01 cm higher than the control. The heaviest main stalk was 27.32 g (CX014) that was 7.25 cm heavier than the control. The lateral stalk height of the 11 DH lines was between 21.30 cm and 18.21 cm, CX013 was significantly shorter than the control; the longest leaf on the lateral stalk was 10.73 cm (CX021) which was significantly longer than the control. The widest leaf on the lateral stalk was 4.71 cm (CX017). The longest petiole of the leaf on the lateral stalk was 3.36 cm (CX021), it was 1.51 cm longer than the control. CX011, CX013, CX017 were significantly wider than the control. The lateral stalk of the DH line (CX015) from Zengcheng flowering Chinese cabbage genotype 17AY012 was shown in Fig. 3A, and the uniformity of the population of DH lines was shown in Fig. 3B.
Table 5 Ploidy identification of regenerated plants of three genotypes of Zengcheng flowering Chinese cabbage. Genotype
No. of evaluated regenerated plants
No. of Haploid plants
No. of Doubled haploid plants
No. of Polyploid plants
Doubling frequency (%)
17AY010 17AY011 17AY012
35 38 55
6 8 12
25 24 34
4 6 9
71.43 63.16 61.82
tillering ability of lateral stalks were shown in Table 6. Some traits were different from the control. Among the 11 DH lines, the maturity period of CX015, CX019, CX020 and CX021 was the shortest that stalks can be harvest after 90 days sowing, while the medium maturity lines CX013,
Table 6 Morphological traits of DH lines obtained from 17AY012 of Zengcheng flowering Chinese cabbage. Material
CX011 CX012 CX013 CX014 CX015 CX016 CX017 CX018 CX019 CX020 CX021 CK
Leaf hair
– + + – – + – – + + – +
Leaf color
dark green light green light green green green light green dark green light green light green light green light green light green
Leaf edge
entire entire entire crenate entire crenate entire entire entire entire entire entire
Leaf shape
oval linear oval oval linear oval oval linear linear linear linear linear
Maturity period
late late medium medium early medium medium late early early early medium
Main stalk
Lateral stalk
Leaf on the lateral stalk
Petiole of the leaf on the lateral stalk
height (cm)
weight (g)
height (cm)
length (cm)
width (cm)
length (cm)
width (cm)
17.82d 21.53b 22.14b 21.50b 21.51b 22.00b 21.48b 19.80c 24.46a 24.38a 21.77b 21.45b
21.20d 15.27i 19.36e 27.32a 25.28b 18.48f 23.97c 20.11e 24.96b 16.56h 17.65g 20.07e
20.03abc 19.42bcd 18.21d 20.80ab 21.07ab 18.96cd 19.60bcd 20.21abc 20.56abc 20.20abc 21.30a 20.20abc
9.52abcd 7.32e 8.80bcde 8.84bcde 9.87ab 8.84bcde 9.71abc 8.22cde 9.30abcd 7.91de 10.73a 8.82bcde
3.30cde 3.31cd 3.94b 3.12cde 2.69e 3.09cde 4.71a 3.06bcd 2.93de 2.78de 3.66bc 2.76de
2.00def 2.22cde 2.90ab 1.82ef 3.14a 2.77abc 2.48bcd 1.57f 1.89def 2.16def 3.36a 1.85ef
1.06a 0.81bc 0.96ab 0.92abc 0.82bc 0.93abc 1.03a 0.79c 0.84bc 0.87bc 0.82bc 0.78c
Note: the data are the average of nine plants. +, hair present; -, hair absent; Means followed by different lowercase letters in the column are significantly different at P = 0.05 level. 61
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Fig. 3. Doubled haploid (DH) lines (CX015) from Zengcheng flowering Chinese cabbage genotype 17AY012. A, single plant of DH lines. B, population of DH lines.
CX020, CX012, CX021) was significantly more than the control. The total stalk weight of CX015 and CX014 reached the maximum of 4655.40 g and 4605.85 g, respectively, and that of other DH lines (CX019, CX012, CX021, CX020, CX013) was heavier than the control, indicating no direct relationship between the total stalk weight and the number of total stalks.
Table 7 Plot yield of DH lines obtained from17AY012 of Zengcheng flowering Chinese cabbage. DH Lines
Average number of total stalks per plant
Average of yield per plant (g)
Plot yield (g)
CX011 CX012 CX013 CX014 CX015 CX016 CX017 CX018 CX019 CX020 CX021 CK
12.33e 25.78c 16.56d 31.67a 32.33a 16.67d 12.78e 13.33e 29.89b 26.44c 25.22c 17.03d
151.54e 284.83b 164.01d 307.06a 310.36a 144.09ef 130.73g 137.65fg 287.06b 272.47c 279.17bc 153.18e
2273.15g 4272.45c 2460.20f 4605.85b 4655.40a 2161.35h 1961.00j 2064.75i 4305.85c 4086.857e 4187.50d 2297.70g
3.4.3. Identification of self-compatibility of the DH lines The self-incompatibility index was calculated for the DH lines from 17AY012. The affinity indexes of florescence were less than 0.3, and the affinity indexes of bud stage were greater than 2. Based on these indexes, the DH lines from 17AY012 lines were selected as self-incompatibility lines in breeding practice (Table 8). 4. Discussion Microspore culture is an effective method of haploid breeding used to produce homozygous DH lines in a short time (Ferrie and Keller, 2004). Until now, microspore culture has been widely used not only in Brassica crops (Sato et al., 1989; Gu et al., 2003, 2014a,b; Zhang et al., 2011), but also in triticale (Eudes and Chugh, 2009), barley (Echavarri et al., 2008), and pepper (Cheng et al., 2013). There are many factors affecting the microspore culture, genotype was one of the most important (Wang et al., 2009; Bhatia et al., 2017). In the present study, we revealed a significant difference (of up to 2.63-fold) in the rates of embryo induction among the three genotypes of Zengcheng flowering Chinese cabbage. This genotype effect on microspore embryogenesis is consistent with previous results (Takahashi et al., 2012). Although microspore embryoids were successfully produced from all the three test materials, the number of embryos could not meet the demand for heterosis breeding. In the process of microspore culture, not all cells can produce normal microspore embryos. Most cells usually die in the early stage of microspore culture, and part of the microspores contribute to the formation of cotyledonary embryos, thereby leading to low rate of embryogenesis (Varnier et al., 2009). Researches showed that the addition of a suitable concentration of plant growth regulators to NLN medium could significantly increase the incidence of microspore embryos (Gland et al., 1988; Sooseong and Ajin, 2000; Jiang and Feng, 2006; Haddadi et al., 2008). Other than plant growth regulators, Asif et al. (2013) reported that organelle antioxidants could also improve microspore embryogenesis. Adding the appropriate amount of antioxidants can maintain normal ROS levels in mitochondria, which is required to promote normal development of the microspores and microspore embryogenesis (Gogvadze et al., 2008; Ganesan and Jayabalan, 2004). In
Note: Means followed by different lowercase letters in the column are significantly different at P = 0.05 level.
Fig. 4. Lateral stalks harvested from CX015 at one time from Zengcheng flowering Chinese cabbage genotype 17AY012.
3.4.2. Plot yield analysis of DH lines According to the plot yield analysis of DH lines from 17AY012 (Table 7), the stalks number per plant of CX015 and CX014 were the most (32). About 10 lateral stalks were harvested each time (Fig. 4), and the average number of total stalks of other DH lines (CX019, 62
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Table 8 Affinity indexes of DH lines obtained from 17AY012 of Zengcheng flowering Chinese cabbage. DH lines
No. of pollinated flowers
No. of Pollinated buds
No. of seeds from flowers
No. of seeds from buds
Compatibility index of florescence
Compatibility index of buds
CX014 CX015 CX019 CX020 CX021
23 16 30 50 20
20 30 32 54 38
5 2 2 11 0
46 72 68 110 83
0.22 0.13 0.07 0.22 0
2.30 2.40 2.13 2.04 2.18
the present study, we found that the addition of 0.2 μM – 5 μM of the organelle antioxidant VcNa can significantly increase the frequency of embryo induction in Zengcheng flowering Chinese cabbages 17AY011 and 17AY012. These concentrations of VcNa added to the NLN-13 medium are possibly in equilibrium with the optimal ranges of ROS concentration required for normal cell metabolism. However, as VcNa concentration exceeded this range, cell viability was obviously reduced during microspore culture, decreasing the rates of embryogenesis and direct conversion to plants. Most experiments conducted in recent years were limited to examining the effects of various stress treatments on microspore embryogenesis in order to establish an efficient microspore protocol (Takahashi et al., 2012; Zhang et al., 2016), and only a few studies further identified and utilized the DH lines (Malik et al., 2008). In our research, plantlets were regenerated directly from embryos or through indirect from callus. The plantlets were rooted and acclimatized to natural environment in growth chambers, and then transferred into the greenhouse until flowering. Their ploidy was checked by flow cytometry, confirming their doubled haploidy. Their characterization in terms of morphological traits and plot yield were identified. Eleven 17AY012 DH lines obtained by microspore culture showed variability in morphology and yield, so that self-imcompatibility lines could be used in the breeding programe. In the present study, we established successfully for the first time microspore culture protocol in Zengcheng flowering Chinese cabbage. The research indicated that organelle antioxidant VcNa can effectively enhance the frequencies of microspore embryogenesis and direct conversion to plants. The rates of spontaneous doubling were greater than 60% in the three genotypes. Moreover, we also obtained excellent DH lines, which laid the foundation of hybrids breeding in Zengcheng flowering Chinese cabbage and will accelerate breeding efficiency. In consideration of the breeding objectives, market demand, and horticultural identification, acquisition of many different types of parent materials will generate more excellent DH lines for heterosis breeding of Zengcheng flowering Chinese cabbage.
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Declarations of interest None. Acknowledgements This work was supported by grants from National Nature Science Foundation of China (31601749) and the National Bulk Vegetable Industry Technical System Vegetable Cell Engineering Breeding Post Expert Special Item (CARS-23-A04). References Asif, M., Eudes, F., Goyal, A., Amundsen, E., Randhawa, H., Spaner, D., 2013. Organelle antioxidants improve microspore embryogenesis in wheat and triticale. In Vitro Cell. Dev. Biol. 49 (5), 489–497. Babbar, S.B., Agarwal, P.K., Sahay, S., Bhojwani, S.S., 2004. Isolated microspore culture of Brassica: an experimental tool for developmental studies and crop improvement. Indian J. Biotechnol. 3, 185–202. Bhatia, R., Dey, S.S., Sood, S., Sharma, K., Parkash, C., Kumar, R., 2017. Efficient
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illuminating and shaking on embryo induction and growth in microspore culture of heading chinese cabbage. J. Korean Soc. Hortic. Sci. 41 (1), 16–20. Takahashi, Y., Yokoi, S., Takahata, Y., 2012. Effects of genotypes and culture conditions on microspore embryogenesis and plant regeneration in several subspecies of Brassica rapa L. Plant Biotechnol. Rep. 4 (6), 297–304. Takahata, Y., Keller, W.A., 1991. High frequency embryogenesis and plant regeneration in isolated microspore culture of Brassica oleracea L. Plant Sci. 74 (2), 235–242. Varnier, A.L., Jacquard, C., Clement, C., 2009. Programmed Cell Death and Microspore Embryogenesis. Springer, Netherlands, pp. 147–154. Vicente, J.G., Dias, J.S., 1996. Production of embryos from microspore cultures of Portuguese tronchuda cabbage landraces. Acta Hortic. Regiotect. 407 (1), 219–226. Wang, T.T., Han, X.L., Zhang, J.H., Bo, O.Y., Lu, Y.G., Ye, Z.B., 2009. Initiation and development of microspore embryogenesis in recalcitrant purple flowering stalk (Brassica campestris ssp. chinensis var. purpurea Hort.) genotypes. Sci. Hortic. 121 (4), 419–424. Winarto, B., Teixeira da Silva, J.A., 2011. Microspore culture protocol for indonesian Brassica oleracea. Plant Cell Tiss. Org. Cult. 107 (2), 305–315. Zeng, A.S., Yan, Y.Y., Yan, J.Y., Song, L.X., Gao, B., Li, J.Q., Hou, X.L., Li, Y., 2015a. Microspore embryogenesis and plant regeneration in Brussels sprouts (Brassica
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Efficient doubled haploid production in microspore culture of Zengcheng flowering Chinese cabbage (Brassica campestris L. ssp. chinensis [L.] Makino var. utilis Tsen et Lee) Liujing Niu1, Fengyan Shi1, Hui Feng, Yun Zhang
T
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Laboratory of Vegetable Genetics Breeding and Biotechnology, Department of Horticulture, Shenyang Agricultural University, No. 120 Dongling Road, Shenhe District, Shenyang, 110866, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Zengcheng flowering Chinese cabbage Microspore culture L-ascorbic acid sodium salt Embryogenesis Doubled haploid lines
Microspore culture has been implemented in breeding programs to produce doubled haploids (DH) and thus accelerate the breeding process. This study aimed to establish an efficient microspore culture protocol for Zengcheng flowering Chinese cabbage, a highly nutritious and valued vegetable, that would be suitable for heterosis breeding. Microspores of three genotypes, 17AY010, 17AY011, and 17AY012 were successfully induced to produce embryos in NLN-13 medium. Two genotypes, 17AY011 and 17AY012 were cultivated in NLN13 medium supplemented with different concentrations (0, 0.2, 1, 5 or 25 μM) of L-ascorbic acid sodium salt (Vitamin C Sodium Salt, VcNa) to enhance microspore embryogenesis and plant regeneration without an intervening callus phase. In both genotypes, the addition of 1 μM and 0.2 μM VcNa significantly increased the frequency of embryogenesis, 6.55-fold and 10.33-fold as compared with the control, respectively. The optimum concentration of VcNa application that would improve the rates of direct plant regeneration in the two tested genotypes was 1 μM. Moreover, the doubled haploid rates of regenerated plants of the three genotypes were above 60%. Five DH lines of 17AY012 with high yield were obtained. According to the measurement of the affinity index, they were prospective self-incompatible lines for hybrid breeding.
1. Introduction Zengcheng flowering Chinese cabbage (Brassica campestris L. ssp. chinensis [L.] Makino var. utilis Tsen et Lee) is a variety of Brassica vegetable crop with edible flower stalk typical for Zengcheng City in Guangdong Province, China. It is appreciated for its fresh, crisp, sweet flavor, and high nutritional value (Liu et al., 2009). As more people are attracted to this variety, its market demand and cultivation area continue to increase steadily. Zengcheng flowering Chinese cabbage is a typical cross-pollinated plant with significant levels of heterosis, and therefore homozygous parental materials play an important role in the breeding of this hybrid. Compared with conventional breeding methods, microspore culture is an effective technology for rapid development of doubled haploid lines, which can significantly accelerate the breeding process (Zhou et al., 2002; Ferrie and Caswell, 2011). Since Lichter (1982) successfully created embryos of Brassica napus L. by using microspore culture, this technique has been used in many plant species. For example, Cao
et al. (1992) produced 200 Chinese cabbage regenerated plants by isolated microspore culture, and the method has been successfully implemented in many other Brassica crops such as broccoli (B. oleracea L. var. italica, Takahata and Keller, 1991; Duijs et al., 1992), tronchuda cabbage (B. oleracea L. var. costata, Vicente and Dias, 1996), ornamental kale (B. oleracea L. var. acephala, Zhang et al., 2008), and sauerkraut cabbage (B. oleracea L. var. capitata, Cao et al., 1990). The induction frequency of microspore-derived embryos is influenced by many factors including donor plant genotype, the developmental stage of microspores, culture conditions, and culture medium (Babbar et al., 2004; Bhowmik et al., 2011; Ferrie and Caswell, 2011; Winarto and Teixeira da Silva, 2011). Many studies have shown that the addition of appropriate amount of chemical reagents to the medium can increase microspore embryogenesis. For instance, adding 1 μM of aminoethoxyvinylglycine to the NLN-13 medium can significantly increase embryo yield of Brussels sprouts (Zeng et al., 2015a), whereas addition of trichostatin A (0.05 μM), suberoylanilide hydroxamic acid (0.05 μM and 0.10 μM), and sodium butyrate (2 μM) effectively increased the
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Corresponding author. E-mail address: [email protected] (Y. Zhang). 1 Liujing Niu and Fengyan Shi contributed equally to this work and share first authorship. https://doi.org/10.1016/j.scienta.2018.09.076 Received 7 June 2018; Received in revised form 26 September 2018; Accepted 27 September 2018 0304-4238/ © 2018 Elsevier B.V. All rights reserved.
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frequency of microspore embryogenesis and plant regeneration in pakchoi (Zhang et al., 2016). In addition, Zeng et al. (2017) reported that the addition of 10 mg/L ascorbate and 20 mg/L glutathione in ½ NLN medium increased the rate of embryo induction of broccoli. Adding organelle antioxidants to the microspore induction medium of Brassica napus and white cabbage allows mitochondria to maintain normal content of reactive oxygen species (ROS), which ensures normal development of microspores, and increases the frequency of microspore-derived embryogenesis (Hoseini et al., 2014; Zeng et al., 2015b). To date, the application of microspore culture in Zengcheng flowering Chinese cabbage has not been reported. Given its limited success in Brassica rapa L., we investigated the effects of different genotypes and addition of organelle antioxidant VcNa on microspore embryogenesis and plant regeneration. The ploidy level of regenerated plants, the morphological characters and plot yield of DH lines were identified. The main purpose of this study is to establish an efficient microspore culture protocol in Zengcheng flowering Chinese cabbage and get DH lines that can be applied to the practice of heterosis breeding.
Table 2 Effect of three genotypes on microspore embryogenesis of Zengcheng flowering Chinese cabbage. Code
No. of embryos per bud ± SD
17AY010 17AY011 17AY012
0.16 ± 0.04b 0.42 ± 0.02a 0.21 ± 0.02b
Note: Means followed by different lowercase letters are significantly different at P = 0.05 level.
supernatant was discarded and the pellet was resuspended in NLN-13 medium (pH 5.84) at a cell density of 1 × 105–2 × 105 microspores·mL−1. The microspore suspension was divided into sterile plastic culture dishes (60 mm × 15 mm), 5 mL per dish, and 100 μL activated carbon solution (0.05 g agarose and 1 g activated carbon suspended in 100 mL double-distilled water) were added to each dish. Finally, the Petri dishes were sealed with Parafilm membranes and incubated at 33 ℃ for 24 h, and then transferred to an incubator, cultured at 25 ℃ in the dark. Once the embryoids were visualized to the naked eye (about 0.5 cm long, about 10 days after microspore culture), the Petri dishes were transferred to a rotary shaker (25 ℃, 50 rpm).
2. Material and methods 2.1. Plant material
2.3. Effect of genotypes on microspore embryogenesis
The heterozygous, self-incompatible donor plants were three genotypes of Zengcheng flowering Chinese cabbage that were imported from Guangdong Province, China. In order to guarantee the uniformity of the microspores for genotype and VcNa treatment, buds from all plants were selected and mixed into groups equally. The codes and basic characteristics of the three genotypes are shown in Table 1. The seeds were sown in the greenhouse at the genetic breeding experimental base of Shenyang Agricultural University in Liaoning Province. When the plants developed four leaves, about 12 cm height, they were transplanted into flowerpots with a diameter of 20 cm and cultivated in the greenhouse under a photoperiod of 16 h and at a day/night temperature of 25/15 °C. Once first flowers developed, the top inflorescences were removed, and after the lateral branches blossomed again, the buds were harvested for microspore culture.
After 21 days, the number of embryos of the three genotypes 17AY010, 17AY011, and 17AY012 was recorded and the difference in microspore embryogenesis among the genotypes was analyzed. 2.4. VcNa treatments Two varieties, 17AY011 and 17AY012, were used for VcNa treatments. The VcNa stock solution with a concentration of 200 μM was dissolved in double-distilled water. The stock solution was filter-sterilized. After dispensing 5 mL microspore suspension into the Petri dish, different volumes of VcNa were added. The final concentration in the NLN-13 medium was 0, 0.2, 1, 5 or 25 μM.
2.2. Microspore culture
2.5. Plant regeneration and ploidy level of the regenerated plants
This experiment followed the microspore culture methods described by Sato et al. (1989) and Hoseini et al. (2014). The pest- and diseasefree inflorescences were harvested on sunny days at 10:00 and pretreated at 4 ℃ for 24 h. Flower buds with the ratio of petal length to anther length of 1/3–4/5 were selected for microspore culture. The buds were sterilized in 75% ethanol (30 s), and 0.1% mercuric chloride solution (6 min) and rinsed with sterilized distilled water three times for 5 min each. The buds were macerated in 10–15 mL B5 liquid medium with 13% (m/v) sucrose, pH 5.84 (Gamborg et al., 1968) using sterile glass rods to release the microspores. The microspores were filtered into a 100 mL beaker through a 74 μm stainless steel cell sieve. The obtained microspore suspension was filtered into 50 mL centrifuge tubes through a 40 μm cell sieve and centrifuged at 2000 rpm for 3 min. The supernatant was removed and 30 mL of B5 medium was added to the pellet and the suspension was centrifuged for additional 3 min. The
The cotyledonary embryos with a length of 3–4 mm from different VcNa concentrations in the NLN medium were inoculated into the conventional solid Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) containing 3% (m/v) sucrose, 0.55% (m/v) agar, and 0.1% (m/v) activated carbon with pH adjusted to 5.84. After 3 weeks of incubation at 25 ± 1 ℃ and 16 h/8 h photoperiod, the rates of embryos directly converting to plants, formation of callus, and mortality were recorded. Flow cytometry was used to identify ploidy of plants regenerated by microspore culture for each genotype (Niu et al., 2015). 2.6. Morphological traits identification and plot yield analysis of DH lines Eleven DH lines of 17AY012 were sown in 32-hole seedling trays on 1 st August 2017, in the plastic tunnel, and after a month they were planted in the field. Complete random design was used with three
Table 1 Donor plants used for microspore embryogenesis of Brassica campestris ssp. Chinensis var. utilis. Genotype code
Variety
Characters
17AY010
sweet Zengcheng flowering Chinese cabbage
17AY011
late-maturing Zengcheng flowering Chinese cabbage
17AY012
late-maturing Zengcheng flowering Chinese cabbage, extrasweet
medium-maturing, fewer lateral tillers, bright green leaf; main stalk height 18–26 cm, main stalk width 2–4 cm late-maturing, more lateral tillers, light green leaf; main stalk height 20–25 cm, main stalk width 2–4 cm late-maturing, more lateral tillers, light green leaf; main stalk height 20–28 cm, main stalk width 2–3 cm
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Fig. 1. Embryo yield of Zengcheng flowering Chinese cabbage genotypes cultivated in NLN-13 medium supplemented with different concentrations of VcNa and embryos development. A, Embryos in the control medium (no VcNa added, 17AY011). B, Embryos in 1 μM VcNa treatment (17AY011). C, Embryos in the control medium (no VcNa added, 17AY012). D, Embryos in 0.2 μM VcNa treatment (17AY012). E, Cotyledonary embryos transferred to MS solid medium. F, regenerated plantlet.
2.7. Self-compatibility test of DH lines
replications. Fifteen plants were planted in each plot with spacing of 30 cm × 60 cm. Leaf hair, leaf color, leaf edge, leaf shape and maturity period were identified. Height and weight of the main stalk, length and width of leaf and petiole on lateral stalk were measured. After the main stalk firstly harvest, lateral stalks were collected every 5 days and collected a total of 3 times. Finally, the total yield of DH plants was calculated.
The DH lines of 17AY012 with high yield were selected to determine the self-compatibility index. After flowering, the plants were bagged 2–3 days before pollination and pollinated artificially at the bud and flowering stage. After 8–10 days, the bags were removed, and the seeds were harvested after ripening. Affinity index was used to select selfincompatible lines, and it was calculated as follows:
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10.33-fold increase when compared with the control) (Fig. 1C and D). In treatments with 25 μM VcNa, the embryo formation rates were inhibited in the two genotypes, but did not reach the significant level (Table 3). The results showed that VcNa concentration of 0.2 μM – 5 μM significantly promoted the microspore embryogenesis in Zengcheng flowering Chinese cabbage, but the optimum concentration differed between the two genotypes. The positive effects of VcNa on plant regeneration in NLN-13 medium were observed in genotypes 17AY011 and 17AY012 (Table 4). The embryos with cotyledons were transferred to MS solid medium (Fig. 1E), and regenerated plants (Fig. 1F) were formed after the embryo development. At 0.2 μM VcNa concentration and compared with the control, 17AY012 increased significantly in terms of the rate of direct conversion to plants (51.02%). In treatments with 1 μM VcNa, 17AY011 had the highest rate of direct conversion to plants (67.67%, 1.33-fold higher compared with the control) and the lowest rate of callus formation (16.24%); a remarkable increase in the rate of direct conversion to plants (52.21%, 1.33-fold higher compared with the control) was observed in 17AY012, whereas the rate of callogenesis decreased to 25.33% in 17AY012. As VcNa concentration increased to 5 μM, the rate of direct conversion to plants was not improved significantly; callus formation were significantly decreased to 23.65% in 17AY011; in 17AY012, the rate of direct conversion to plants increased to 49.00%, which was significantly different compared to the control, callus formation rate decreased to 20.07%. There was no significant difference among 0.2, 1 and 5 μM at the rates of direct conversion to plants and callus formation in 17AY012. Finally, at the highest tested VcNa concentration of 25 μM, the rate of direct conversion to plants in both genotypes was decreased, but there was no significant difference compared to the control; the mortality was increased significantly in both genotypes. To sum up, the optimal concentration of VcNa in the NLN-13 medium that would increase the rate of direct conversion to plants and reduce the formation of callus in the two genotypes was 1 μM.
Table 3 Effect of four concentrations of L-ascorbic acid sodium salt (VcNA) on microspore embryogenesis of two genotypes of Zengcheng flowering Chinese cabbage. VcNa (μM)
No. of embryos per bud ± SD
0 0.2 1 5 25
17AY011
17AY012
0.42 1.67 2.75 1.29 0.29
0.21 2.17 0.83 1.54 0.17
± ± ± ± ±
0.08c 0.17b 0.18a 0.15b 0.08c
± ± ± ± ±
0.08d 0.17a 0.11c 0.15b 0.05d
Note: Means followed by different lowercase letters in the column are significantly different at P = 0.05 level.
Affinity index = number of self-pollinated seeds/number of pollinated flowers (buds). 2.8. Experimental design and data analysis The microspore embryogenesis experiments were conducted using complete randomized design with three replicates (each replicate comprised six Petri dishes, each Petri dish contained 5 buds). Data were analyzed using SPSS (16.0) and the means were compared by using Duncan’s multiple range test at P = 0.05. 3. Results 3.1. Effect of genotypes on microspore embryogenesis in Zengcheng flowering Chinese cabbage Embryos of all three genotypes were successfully induced from microspore cultures using NLN-13 medium, but the frequency of microspore embryogenesis was low. The highest embryo induction rate was observed in 17AY011 (0.42 embryos per bud) and the lowest in 17AY010 (0.16 embryos per bud) (Table 2). There was a significant difference in the frequency of microspore embryogenesis between 17AY011 and the other two different genotypes.
3.3. Ploidy identification of regenerated plants in Zengcheng flowering Chinese cabbage The ploidy of regenerated plants was identified by flow cytometry. The histogram peaks near 100 (Fig. 2A), 200 (Fig. 2B), and 400 (Fig. 2C) indicated that the plant was haploid, doubled haploid, and tetraploid, respectively. The rates of doubling frequency of the three genotypes were more than 60%, and the highest DH rate was 71.43% (17AY010) (Table 5).
3.2. Effect of VcNa on microspore embryogenesis and plant regeneration We selected 17AY011 and 17AY012 to examine the effects of different concentrations of VcNa on the frequency of microspore embryogenesis. In 17AY011, when the VcNa at the concentration of 0.2 μM and 5 μM, the embryo induction rate increased to 1.67 and 1.29 embryos per bud, respectively. The highest frequency of microspore embryogenesis, 2.75 embryos per bud, was observed in NLN-13 medium with 1 μM VcNa; this was 6.55-fold higher than that of the control (Fig. 1A and B). In genotype 17AY012, the highest frequency of embryogenesis was obtained at 0.2 μM VcNa (2.17 embryos per bud, a
3.4. Identification of DH lines 3.4.1. Morphological traits identification of DH lines The plant morphological traits of the DH lines from 17AY012 such as presence or absence of hairs, leaf traits, maturing period, and
Table 4 The effect of VcNa on plant regeneration of two genotypes of Zengcheng flowering Chinese cabbage. Genotype
17AY011
17AY012
Concentration (μM)
Rate of embryos directly converting to plants (%)
Rate of embryos converting to callus (%)
Rate of embryos death (%)
0 0.2 1 5 25 0 0.2 1 5 25
51.00 47.67 67.67 55.34 41.67 30.67 51.02 52.21 49.00 22.33
33.23 37.33 16.24 23.65 21.00 44.30 22.35 25.33 20.07 35.62
15.77 15.00 16.09 21.01 37.33 25.03 26.63 22.46 30.93 42.05
± ± ± ± ± ± ± ± ± ±
8.23b 1.47b 3.11a 5.38ab 6.34b 3.16b 1.74a 5.37a 4.78a 4.72b
± ± ± ± ± ± ± ± ± ±
4.63a 2.66a 2.28b 4.57b 2.04b 3.62a 2.37c 3.36c 3.53c 1.48b
Note: Means followed by different lowercase letters within the genotype in the column are significantly different at P = 0.05 level. 60
± ± ± ± ± ± ± ± ± ±
6.26b 1.37b 2.25b 3.16b 1.72a 4.21b 1.07b 2.66b 5.73ab 3.42a
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Fig. 2. DNA content distribution in leaves of regenerated plants. A, Haploid. B, Doubled haploid. C, Polyploid.
CX014, CX016 and CX017 needed 97 days to be harvest that was the same with the control, the DH lines CX011, CX012 and CX018 were 110 days- maturity which was the latest. The highest main stalk was 24.46 cm (CX019) that was 3.01 cm higher than the control. The heaviest main stalk was 27.32 g (CX014) that was 7.25 cm heavier than the control. The lateral stalk height of the 11 DH lines was between 21.30 cm and 18.21 cm, CX013 was significantly shorter than the control; the longest leaf on the lateral stalk was 10.73 cm (CX021) which was significantly longer than the control. The widest leaf on the lateral stalk was 4.71 cm (CX017). The longest petiole of the leaf on the lateral stalk was 3.36 cm (CX021), it was 1.51 cm longer than the control. CX011, CX013, CX017 were significantly wider than the control. The lateral stalk of the DH line (CX015) from Zengcheng flowering Chinese cabbage genotype 17AY012 was shown in Fig. 3A, and the uniformity of the population of DH lines was shown in Fig. 3B.
Table 5 Ploidy identification of regenerated plants of three genotypes of Zengcheng flowering Chinese cabbage. Genotype
No. of evaluated regenerated plants
No. of Haploid plants
No. of Doubled haploid plants
No. of Polyploid plants
Doubling frequency (%)
17AY010 17AY011 17AY012
35 38 55
6 8 12
25 24 34
4 6 9
71.43 63.16 61.82
tillering ability of lateral stalks were shown in Table 6. Some traits were different from the control. Among the 11 DH lines, the maturity period of CX015, CX019, CX020 and CX021 was the shortest that stalks can be harvest after 90 days sowing, while the medium maturity lines CX013,
Table 6 Morphological traits of DH lines obtained from 17AY012 of Zengcheng flowering Chinese cabbage. Material
CX011 CX012 CX013 CX014 CX015 CX016 CX017 CX018 CX019 CX020 CX021 CK
Leaf hair
– + + – – + – – + + – +
Leaf color
dark green light green light green green green light green dark green light green light green light green light green light green
Leaf edge
entire entire entire crenate entire crenate entire entire entire entire entire entire
Leaf shape
oval linear oval oval linear oval oval linear linear linear linear linear
Maturity period
late late medium medium early medium medium late early early early medium
Main stalk
Lateral stalk
Leaf on the lateral stalk
Petiole of the leaf on the lateral stalk
height (cm)
weight (g)
height (cm)
length (cm)
width (cm)
length (cm)
width (cm)
17.82d 21.53b 22.14b 21.50b 21.51b 22.00b 21.48b 19.80c 24.46a 24.38a 21.77b 21.45b
21.20d 15.27i 19.36e 27.32a 25.28b 18.48f 23.97c 20.11e 24.96b 16.56h 17.65g 20.07e
20.03abc 19.42bcd 18.21d 20.80ab 21.07ab 18.96cd 19.60bcd 20.21abc 20.56abc 20.20abc 21.30a 20.20abc
9.52abcd 7.32e 8.80bcde 8.84bcde 9.87ab 8.84bcde 9.71abc 8.22cde 9.30abcd 7.91de 10.73a 8.82bcde
3.30cde 3.31cd 3.94b 3.12cde 2.69e 3.09cde 4.71a 3.06bcd 2.93de 2.78de 3.66bc 2.76de
2.00def 2.22cde 2.90ab 1.82ef 3.14a 2.77abc 2.48bcd 1.57f 1.89def 2.16def 3.36a 1.85ef
1.06a 0.81bc 0.96ab 0.92abc 0.82bc 0.93abc 1.03a 0.79c 0.84bc 0.87bc 0.82bc 0.78c
Note: the data are the average of nine plants. +, hair present; -, hair absent; Means followed by different lowercase letters in the column are significantly different at P = 0.05 level. 61
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Fig. 3. Doubled haploid (DH) lines (CX015) from Zengcheng flowering Chinese cabbage genotype 17AY012. A, single plant of DH lines. B, population of DH lines.
CX020, CX012, CX021) was significantly more than the control. The total stalk weight of CX015 and CX014 reached the maximum of 4655.40 g and 4605.85 g, respectively, and that of other DH lines (CX019, CX012, CX021, CX020, CX013) was heavier than the control, indicating no direct relationship between the total stalk weight and the number of total stalks.
Table 7 Plot yield of DH lines obtained from17AY012 of Zengcheng flowering Chinese cabbage. DH Lines
Average number of total stalks per plant
Average of yield per plant (g)
Plot yield (g)
CX011 CX012 CX013 CX014 CX015 CX016 CX017 CX018 CX019 CX020 CX021 CK
12.33e 25.78c 16.56d 31.67a 32.33a 16.67d 12.78e 13.33e 29.89b 26.44c 25.22c 17.03d
151.54e 284.83b 164.01d 307.06a 310.36a 144.09ef 130.73g 137.65fg 287.06b 272.47c 279.17bc 153.18e
2273.15g 4272.45c 2460.20f 4605.85b 4655.40a 2161.35h 1961.00j 2064.75i 4305.85c 4086.857e 4187.50d 2297.70g
3.4.3. Identification of self-compatibility of the DH lines The self-incompatibility index was calculated for the DH lines from 17AY012. The affinity indexes of florescence were less than 0.3, and the affinity indexes of bud stage were greater than 2. Based on these indexes, the DH lines from 17AY012 lines were selected as self-incompatibility lines in breeding practice (Table 8). 4. Discussion Microspore culture is an effective method of haploid breeding used to produce homozygous DH lines in a short time (Ferrie and Keller, 2004). Until now, microspore culture has been widely used not only in Brassica crops (Sato et al., 1989; Gu et al., 2003, 2014a,b; Zhang et al., 2011), but also in triticale (Eudes and Chugh, 2009), barley (Echavarri et al., 2008), and pepper (Cheng et al., 2013). There are many factors affecting the microspore culture, genotype was one of the most important (Wang et al., 2009; Bhatia et al., 2017). In the present study, we revealed a significant difference (of up to 2.63-fold) in the rates of embryo induction among the three genotypes of Zengcheng flowering Chinese cabbage. This genotype effect on microspore embryogenesis is consistent with previous results (Takahashi et al., 2012). Although microspore embryoids were successfully produced from all the three test materials, the number of embryos could not meet the demand for heterosis breeding. In the process of microspore culture, not all cells can produce normal microspore embryos. Most cells usually die in the early stage of microspore culture, and part of the microspores contribute to the formation of cotyledonary embryos, thereby leading to low rate of embryogenesis (Varnier et al., 2009). Researches showed that the addition of a suitable concentration of plant growth regulators to NLN medium could significantly increase the incidence of microspore embryos (Gland et al., 1988; Sooseong and Ajin, 2000; Jiang and Feng, 2006; Haddadi et al., 2008). Other than plant growth regulators, Asif et al. (2013) reported that organelle antioxidants could also improve microspore embryogenesis. Adding the appropriate amount of antioxidants can maintain normal ROS levels in mitochondria, which is required to promote normal development of the microspores and microspore embryogenesis (Gogvadze et al., 2008; Ganesan and Jayabalan, 2004). In
Note: Means followed by different lowercase letters in the column are significantly different at P = 0.05 level.
Fig. 4. Lateral stalks harvested from CX015 at one time from Zengcheng flowering Chinese cabbage genotype 17AY012.
3.4.2. Plot yield analysis of DH lines According to the plot yield analysis of DH lines from 17AY012 (Table 7), the stalks number per plant of CX015 and CX014 were the most (32). About 10 lateral stalks were harvested each time (Fig. 4), and the average number of total stalks of other DH lines (CX019, 62
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Table 8 Affinity indexes of DH lines obtained from 17AY012 of Zengcheng flowering Chinese cabbage. DH lines
No. of pollinated flowers
No. of Pollinated buds
No. of seeds from flowers
No. of seeds from buds
Compatibility index of florescence
Compatibility index of buds
CX014 CX015 CX019 CX020 CX021
23 16 30 50 20
20 30 32 54 38
5 2 2 11 0
46 72 68 110 83
0.22 0.13 0.07 0.22 0
2.30 2.40 2.13 2.04 2.18
the present study, we found that the addition of 0.2 μM – 5 μM of the organelle antioxidant VcNa can significantly increase the frequency of embryo induction in Zengcheng flowering Chinese cabbages 17AY011 and 17AY012. These concentrations of VcNa added to the NLN-13 medium are possibly in equilibrium with the optimal ranges of ROS concentration required for normal cell metabolism. However, as VcNa concentration exceeded this range, cell viability was obviously reduced during microspore culture, decreasing the rates of embryogenesis and direct conversion to plants. Most experiments conducted in recent years were limited to examining the effects of various stress treatments on microspore embryogenesis in order to establish an efficient microspore protocol (Takahashi et al., 2012; Zhang et al., 2016), and only a few studies further identified and utilized the DH lines (Malik et al., 2008). In our research, plantlets were regenerated directly from embryos or through indirect from callus. The plantlets were rooted and acclimatized to natural environment in growth chambers, and then transferred into the greenhouse until flowering. Their ploidy was checked by flow cytometry, confirming their doubled haploidy. Their characterization in terms of morphological traits and plot yield were identified. Eleven 17AY012 DH lines obtained by microspore culture showed variability in morphology and yield, so that self-imcompatibility lines could be used in the breeding programe. In the present study, we established successfully for the first time microspore culture protocol in Zengcheng flowering Chinese cabbage. The research indicated that organelle antioxidant VcNa can effectively enhance the frequencies of microspore embryogenesis and direct conversion to plants. The rates of spontaneous doubling were greater than 60% in the three genotypes. Moreover, we also obtained excellent DH lines, which laid the foundation of hybrids breeding in Zengcheng flowering Chinese cabbage and will accelerate breeding efficiency. In consideration of the breeding objectives, market demand, and horticultural identification, acquisition of many different types of parent materials will generate more excellent DH lines for heterosis breeding of Zengcheng flowering Chinese cabbage.
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