Breeding Science
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Research Papers
Efficient haploid and doubled haploid production from unfertilized ovule culture of gentians (Gentiana spp.)
Hisako Doi, Nobue Hoshi, Eri Yamada, Shuji Yokoi, Masahiro Nishihara, Takashi Hikage, Yoshihito Takahata
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Keywords: Gentiana triflora, G. scabra, unfertilized ovule culture, genotype, haploid, doubled haploid
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2013 Volume 63 Issue 4 Pages 400-406

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Abstract

Factors affecting reliable plant regeneration from unfertilized ovule culture of gentians (Gentiana spp.) were examined. Cold pretreatment (4°C) of flower buds enhanced or maintained production of embryo-like structure (ELS). When 43 genotypes were surveyed in two different labs, 40 of them produced ELSs ranging from 0.01 to 26.5 ELSs per flower bud. No ELSs could be obtained in three genotypes. A significant correlation (r = 0.64) was observed between the number of ELS per flower and the frequency of responding flower buds. Eight genotypes of G. triflora, which were used as common materials in two different labs, produced ELSs in both labs. The ploidy levels of a total of 1,515 regenerated plantlets were determined, revealing that the majority of these plants consisted of haploids (57.9%) and diploids (34.3%). However, the frequency of haploids and diploids was different between G. triflora and G. scabra, and G. triflora showed higher frequencies of haploids than G. scabra. When haploids were treated with oryzalin for chromosome doubling, diploids and tetraploids were obtained. These results demonstrate that the unfertilized ovule culture technique of gentians is a powerful tool for obtaining haploids and DHs because of its reproducible and reliable nature and application to a wide range of genotypes.

Introduction

Some species of Gentiana have been used as ornamental plants in Europe and Asia. Although gentians are commonly used in rock gardens and garden borders in Europe, they are one of the most important plants for cut-flower and pot-plant use in Japan. Both G. triflora and G. scabra, which grow wild in Japan, have been cultivated commercially as ornamental plants and many F1 and clonal cultivars have been bred (Takahata et al. 1995, Yoshiike 1992). Although homozygous parental lines are indispensable for F1 hybrid breeding, it is difficult to obtain homozygous lines owing to their intense inbreeding depression.

Recently, successful production of haploids and doubled haploids (DHs) has been reported in male and female gametophytic cells by in vitro culture of anthers (androgenesis) in G. triflora and unfertilized ovules (gynogenesis) in some species of Gentiana (G. triflora, G. scabra and their hybrids), respectively (Doi et al. 2010, 2011, Pathirana et al. 2011). Doi et al. (2011) indicated that unfertilized ovule culture has more advantages than anther culture from the point of view of embryogenesis efficiency, limited influence of donor plant genotype and a high frequency of haploids and DHs production. However, this conclusion was inferred from results derived from limited plant materials (four genotypes). For practical use of unfertilized ovule culture for breeding, it is important to determine whether this technique can be applied to a number of genotypes and used with high reproducibility in other labs.

Cold pretreatment of flower buds or inflorescences before culture has been reported to enhance the frequency of embryogenesis not only through androgenesis but also through gynogenesis in several species (Chen et al. 2011, Sopory and Munshi 1996). In addition, the storage of buds in a low temperature for certain periods can avoid the concentration of culture work over a limited time (Sato et al. 2002, Takahashi et al. 2012).

In this study, to clarify the genotypic variation in embryo-like structure (ELS) production from unfertilized ovule culture of gentians and the reproducibility of the results obtained by using this technique, we investigated the ability of ELS production and plant regeneration on 43 genotypes at two different labs. In addition, the effect of cold pre-treatment on unfertilized ovule culture and the optimum condition of chromosome doubling of haploid plants were examined.

Materials and Methods

Plant materials

Forty-three genotypes of gentians, which are listed in Table 1, were used in this study. They consisted of 12 cultivars and 31 lines of G. triflora (2n = 26), G. scabra (2n = 26), G. triflora var. japonica f. montana (2n = 26) and their hybrids (G. triflora × G. scabra, G. scabra × G. triflora, G. triflora var. japonica f. montana × G. triflora). These materials were grown in experimental fields and in a greenhouse at Hachimantai City Floricultural Research and Development Center, Hachimantai, Iwate, Japan and at Iwate Agricultural Research Center (IARC), Kitakami, Iwate, Japan.

Table 1 List of cultivars/lines used in this study
Species
 Cultivar/line		 Type Source
G. triflora
 Ashiro-no-aki F1 Hybrid Hachimantai
 Cust F1 Hybrid Kitakami
 Giovanni F1 Hybrid Kitakami
 Goku-wase2 F1 Hybrid Kitakami
 Homoi F1 Hybrid Kitakami
 Ihatovo F1 Hybrid Kitakami
 Iwate F1 Hybrid Kitakami
 Marjel F1 Hybrid Kitakami
 Maciry F1 Hybrid Kitakami
 06-6 Sib-cross line Kitakami
 06-8 Sib-cross line Kitakami
 05-01B Sib-cross line Kitakami
 05-02B Sib-cross line Kitakami
 05-03B Sib-cross line Kitakami
 05-04B Sib-cross line Kitakami
 05-05B Sib-cross line Kitakami
 05-07B Sib-cross line Kitakami
 05-12B Sib-cross line Kitakami
 05-17B Sib-cross line Kitakami
 05-571 Sib-cross line Kitakami
 06-512YRy Sib-cross line Kitakami
 06-65SPB Sib-cross line Kitakami
 07-520 Sib-cross line Kitakami
 07-533 Sib-cross line Kitakami
G. scabra
 17-260 F1 Hybrid Hachimantai
 Alta F1 Hybrid Kitakami
G. triflora var. japonica f. montana
 AZ1 early1 Clonal line Hachimantai
G. triflora × G. scabra
 14-218-13 Clonal line Hachimantai
 14-218-14 Clonal line Hachimantai
 14-218-20 Clonal line Hachimantai
 14-218-21 Clonal line Hachimantai
 14-218-3 Clonal line Hachimantai
 14-218-30 Clonal line Hachimantai
 17-386 F1 Hybrid Hachimantai
 17-488 F1 Hybrid Hachimantai
G. scabra × G. triflora
 LBbc F1 Hybrid Kitakami
 Polarno blue F1 Hybrid Kitakami
 07-119B Sib-cross line Kitakami
 07-119P Sib-cross line Kitakami
 07-119W Sib-cross line Kitakami
 07-123B Sib-cross line Kitakami
 07-123P Sib-cross line Kitakami
G. triflora var. japonica f. montana × G. triflora
 17-771 F1 Hybrid Hachimantai

Unfertilized ovule culture and plantlet regeneration

Unfertilized ovule culture was carried out as described by Doi et al. (2011) at two different labs, Iwate University, Morioka and IARC, Kitakami. Flower buds, which were in developmental stage 4 to stage 6 (Doi et al. 2011), were taken from the donor plants. After the petals and stamens were removed, the pistils were surface-sterilized in 70% ethanol for 30 s, followed by sodium hypochlorite solution (2% active chlorine) for 15 min and then rinsed three times with sterile distilled water (5 min each time). Ovules excised from a pistil were cultured on a 60-mm plastic Petri dish containing 0.8% agar-solidified 1/2 NLN medium (Takahata and Keller 1991) supplemented with 10% sucrose. The Petri dishes were maintained at 25°C under dark condition. There were at least three plates per experiment and each experiment had at least three independent replicates. Statistical analysis was performed using the computer program JMP 8.0 (SAS Institute Inc.).

The ELSs developed from ovules were transferred to modified agar (1.0%)-solidified MS medium (Murashige and Skoog 1962) with the concentration of major salts reduced by 50% (1/2MS) and supplemented with 3% sucrose and 1.0 mg/l GA3 and incubated at 20°C with a 16 h/day photoperiod. Regenerated plants were grown in vermiculate and then transferred to soil in a greenhouse.

Cold pretreatment

The effect of cold pretreatment for unfertilized ovule culture was investigated using four genotypes (G. triflora cv. Ashiro-no-Aki, G. scabra line 17-260, G. triflora × G. scabra lines 17-386 and 17-488), which already demonstrated the ability of ELS production (Doi et al. 2011). After anthers were removed from each flower, inflorescences were stored at low temperature (4°C) for 3, 7 and 14 days in the dark. After this treatment, unfertilized ovules were cultured as described above.

Ploidy level determination by flow cytometry

The ploidy level of regenerated plantlets was analyzed using Partec CyFlow PA (Partec GmbH, Germany) and Cell Lab Quanta SC (Beckman Coulter, USA) at Iwate University and IARC, respectively. Materials for ploidy determination were prepared from young leaf (approx. 25 mm2) of regenerated plants. Sample preparation and measurement of the ploidy level were performed according to Doi et al. (2010) in the former analysis and Mishiba et al. (2009) in the latter.

Chromosome doubling treatment

Chromosome doubling of haploids was performed as described by Morgan et al. (2003) with minor modification. Haploid plants were maintained in 1/2 MS medium, and subcultured to propagation (Pr) medium (1/2MS medium supplemented with 1.0 mg/l BA and 1.0 mg/l GA3) to increase shoot numbers for chromosome doubling treatment. After 4 weeks of culture, elongated shoots containing axillary buds were cut and subcultured to Pr medium containing 50 μM oryzalin for 1, 2, 3 and 4 weeks. Their explants were then cultured in Pr medium for 6 weeks and elongated axillary shoots were transferred to Pr medium without BA. After 4 weeks of culture, normal plantlets were subcultured to 1/2MS medium and their ploidy level was determined by flow cytometry.

Results

Effect of cold pretreatment

The effect of cold pretreatment of plant materials on ELS production was examined using four genotypes. Cold pre-treatment produced more ELSs than non-treatment in three genotypes except for G. triflora × G. scabra line 17-386 (Table 2). In particular, cold pretreatment for 7 and 14 days tended to exhibit a higher response, and a significant increase was shown in G. triflora cv. Ashiro-no-Aki treated at low temperature for 7 days.

Table 2 Effect of cold pretreatment (4°C) on ELS production in unfertilized ovule culture of gentians
Species
 Cultivar/line		 Cold pretreatment (day) No. of cultured flower buds Frequency of responding flower buds (%) ELS induction
No. of ELS ELS per flower bud*
G. triflora
 cv. Ashiro-no-Aki		 0 64 62.6 106 1.66 b
3 88 66.2 162 1.84 b
7 83 73.5 570 6.87 a
14 102 53.7 210 2.06 b
G. scabra
 17-260		 0 56 41.7 53 0.95 b
3 57 60.5 107 1.88 b
7 66 65.6 107 1.62 b
14 58 56.0 111 1.91 b
G. triflora × G. scabra
 17-386		 0 76 7.2 8 0.11 b
3 66 5.5 6 0.09 b
7 66 0.0 0 0.00 b
14 85 5.2 4 0.05 b
 17-488 0 83 67.2 111 1.34 b
3 83 57.3 95 1.14 b
7 89 68.2 177 1.99 b
14 98 78.9 195 1.99 b
*  Means within columns followed by different letters are different at the 0.05 level by the Tukey-Kramer’s test.

Effects of genotypes and labs

The effect of genotypes on production of ELSs from unfertilized ovule culture was examined independently at Iwate Univ. and IARC and the results are shown in Tables 3, 4, respectively. Although genotypic variations on ELS production are shown, almost all genotypes produced ELSs in both labs except for 3 genotypes. The plant materials used at Iwate Univ. were treated at low temperature for 7–14 days and those used at IARC were not treated at low temperature. All 23 genotypes used at Iwate Univ. produced ELSs ranging from 26.5 ELSs per flower bud of G. triflora cv. Ihatovo to 0.03 of G. triflora × G. scabra line 17-386 (Table 3). In IARC, of 28 genotypes used, 25 produced ELSs ranging from 20.6 ELSs per flower bud of G. triflora line 06-6 to 0.01 of G. scabra × G. triflora line 07-123B (Table 4). Three genotypes, G. triflora 05-12B, G. tiflora × G. scabra LBbc and Polarno blue, produced no ELSs. The frequency of responding flower buds was significantly related to the number of ELS per flower bud (r = 0.64 based on merged data of Tables 3, 4).

Table 3 Effect of genotypes on ELS production in unfertilized ovule culture of gentians at Iwate University
Species Cultivar/line No. of cultured flower buds Frequency of responding flower buds (%) ELSs production
No. of ELS ELS per flower bud*
G. triflora Ashiro-no-Aki 185 63.6 780 4.22 cd
Cust 112 34.6 331 2.96 cd
Ihatovo 58 85.6 1539 26.53 a
Marjel 121 56.5 438 3.62 cd
Maciry 112 59.1 643 5.74 bcd
06-6 160 68.6 1007 6.29 bcd
05-01B 44 20.6 34 0.77 cd
05-02B 62 57.6 90 1.45 cd
05-03B 93 52.0 196 2.11 cd
05-04B 52 44.1 80 1.54 cd
05-05B 66 94.1 697 10.56 bc
05-07B 48 50.0 73 1.52 cd
G. scabra 17-260 124 60.8 218 1.76 d
G. triflora var. japonica f. montana AZ1 early1 110 6.2 14 0.13 cd
G. triflora × G. scabra 17-386 151 2.6 4 0.03 d
17-488 187 73.5 372 1.99 cd
14-218-3 99 23.1 24 0.24 d
14-218-13 79 19.9 17 0.22 d
14-218-14 78 46.7 72 0.92 d
14-218-20 70 66.3 138 1.97 cd
14-218-21 101 61.9 139 1.38 d
14-218-30 81 70.0 218 2.69 cd
G. triflora var. japonica f. montana × G. triflora 17-771 122 87.1 1485 12.17 b
*  Means within columns followed by the different letters are significantly different at the 0.05 level by Tukey-Kramer’s test.

The statistical analysis was carried out on the whole data set.

Table 4 Effect of genotypes on ELS production in unfertilized ovule culture of gentians at Iwate Agricultural Research Center (IARC)
Species Cultivar/line No. of cultured flower buds Frequency of responding flower buds (%) ELS production
No. of ELSs ELSs per flower bud*
G. triflora Cust 262 36.3 293 1.12 f
Giovanni 42 52.4 73 1.74 def
Goku-wase2 233 73.8 1015 4.36 b
Homoi 11 63.6 19 1.73 bcdef
Ihatovo 97 41.2 161 1.66 ef
Iwate 33 27.3 11 0.33 ef
Marjel 136 6.6 17 0.13 f
Maciry 130 38.5 139 1.07 ef
06-6 22 68.2 112 5.09 bcd
06-8 7 71.4 144 20.57 a
05-04B 25 16.0 4 0.16 ef
05-05B 57 26.3 23 0.40 f
05-07B 63 9.5 6 0.10 f
05-12B 14 0.0 0 0.00 ef
05-17B 107 22.4 26 0.24 f
05-571 80 7.5 6 0.08 f
06-512YRy 106 70.8 286 2.70 cde
06-65SPB 94 18.1 27 0.29 f
07-520 112 25.9 53 0.47 f
07-533 114 66.7 508 4.46 bc
G. scabra Alta 86 16.3 75 0.87 ef
G. triflora × G. scabra LBbc 75 0.0 0 0.00 f
Polarno blue 134 0.0 0 0.00 f
G. scabra × G. triflora 07-119B 67 40.3 57 0.85 ef
07-119P 48 52.1 54 1.13 ef
07-119W 56 17.9 13 0.23 f
07-123B 97 1.0 1 0.01 f
07-123P 99 16.2 53 0.54 f
*  Means within columns followed by different letters are significantly different at the 0.05 level by the Tukey-Kramer’s HSD test.

The statistical analysis was carried out on the whole data set.

Eight genotypes of G. triflora cvs./lines, i.e., Cust, Ihatovo, Marjel, Maciry, 06-6, 05-04B, 05-05B and 05-07B, which were used as common materials in two different labs, produced ELSs in both labs. However, the frequency of ELS production in Iwate Univ. (a mean value of 7.35 ELSs per flower) was higher than those in IARC (1.22 ELSs per flower). Such a trend was also found in results obtained from data of all genotypes (a mean value of 3.95 ELSs per flower in the former vs. 1.80 ELSs per flower in the latter).

Ploidy level of regenerated plants

The ploidy levels of a total of 1,515 regenerated plantlets, which were randomly chosen from regenerated plantlets, were determined by flow cytometry (Table 5). The majority of these plants consisted of haploids (57.9%) and diploids (34.3%). A higher frequency of haploids was obtained in G. triflora (66.4% of haploids and 28.1% of diploids), while a higher frequency of diploids was obtained in G. scabra (24.9% of haploids and 59.8% of diploids). Besides diploid and haploid, a higher ploidy from triploid to hexaploid and chimera were found in the several genotypes.

Table 5 Determination of ploidy level of plantlets derived from unfertilized ovule culture of gentian
Cultivar/line Number of analyzed plants No. of plants (%)
x 2x 3x 4x 5x 6x Chimeric plant
G. triflora
 Ashirono-aki 70 36 (51.4) 27 (38.6) 3 (4.3) 0 0 2 (2.9) 2 (2.9)
 Cust 53 36 (67.9) 15 (28.3) 0 1 (1.9) 0 0 1 (1.9)
 Giovanni 19 14 (73.7) 5 (26.3) 0 0 0 0 0
 Goku-wase2 289 170 (58.8) 112 (38.8) 3 (1.0) 4 (1.4) 0 0 0
 Homoi 5 5 (100) 0 0 0 0 0 0
 Ihatovo 87 53 (60.9) 21 (24.1) 4 (4.6) 4 (4.6) 0 0 5 (5.7)
 Iwate 1 1 (100) 0 0 0 0 0 0
 Marjel 35 17 (48.6) 14 (40) 1 (2.9) 2 (5.7) 0 0 1 (2.9)
 Maciry 107 68 (63.7) 31 (29.0) 0 3 (2.8) 0 0 5 (4.7)
 06-6 99 57 (57.6) 33 (33.3) 5 (5.1) 3 (3.0) 0 0 1 (1.0)
 06-8 25 17 (68.0) 7 (28.0) 1 (4.0) 0 0 0 0
 05-01B 7 4 (57.1) 1 (14.3) 0 0 0 0 2 (28.6)
 05-02B 9 7 (77.8) 2 (22.2) 0 0 0 0 0
 05-03B 55 36 (65.5) 17 (30.9) 1 (1.8) 1 (1.8) 0 0 0
 05-04B 19 10 (52.6) 8 (42.1) 0 0 0 0 1 (5.3)
 05-05B 16 8 (50.0) 7 (43.8) 1 (6.2) 0 0 0 0
 05-07B 10 3 (30.0) 6 (60.0) 0 0 0 0 1 (10.0)
 05-17B 2 2 (100) 0 0 0 0 0 0
 06-512Yry 96 91 (94.8) 4 (4.2) 1 (1.0) 0 0 0 0
 06-65SPB 5 5 (100) 0 0 0 0 0 0
 07-520 7 6 (85.7) 1 (14.3) 0 0 0 0 0
 07-533 128 114 (89.1) 11 (8.6) 2 (1.6) 1 (0.8) 0 0 0
 Total 1144 760 (66.4) 322 (28.1) 22 (1.9) 19 (1.7) 0 2 (0.2) 19 (1.7)
G. scabra
 17-260 176 42 (23.9) 102 (58) 11 (6.3) 16 (9.1) 0 0 5 (2.8)
 Alta 33 10 (30.3) 23 (69.7) 0 0 0 0 0
 Total 209 52 (24.9) 125 (59.8) 11 (5.3) 16 (7.7) 0 0 5 (2.4)
G. triflora var. japonica f. montana
 AZ1 early1 1 0 1 (100) 0 0 0 0 0
G. triflora × G. scabra
 14-218-20 4 2 (50.0) 2 (50.0) 0 0 0 0 0
 14-218-21 9 1 (11.1) 7 (77.8) 0 0 0 0 1 (11.1)
 14-218-30 2 0 2 (100) 0 0 0 0 0
 17-386 3 1 (33.3) 2 (66.7) 0 0 0 0 0
 17-488 37 7 (18.9) 28 (75.7) 1 (2.7) 1 (2.7) 0 0 0
 Total 55 11 (20) 41 (74.5) 1 (1.8) 1 (1.8) 0 0 1 (1.8)
G. scabra × G. triflora
 07-119B 19 10 (52.6) 6 (31.6) 1 (5.3) 1 (5.3) 1 (5.3) 0 0
 07-119P 8 5 (62.5) 3 (37.5) 0 0 0 0 0
 07-119W 1 0 1 (100) 0 0 0 0 0
 Total 28 15 (53.6) 10 (35.7) 1 (3.6) 1 (3.6) 1 (3.6) 0 0
G. triflora var. japonica f. Montana × G. triflora
 17-771 78 39 (50.0) 25 (32.1) 9 (11.5) 2 (2.6) 0 0 3 (3.8)
 Total 1515 877 (57.9) 524 (34.6) 44 (2.9) 39 (2.6) 1 (0.06) 2 (13.2) 28 (1.85)

Chromosome doubling treatment

In order to obtain DH plants effectively, haploids of G. triflora, G. scabra and G. triflora var. japonica f. montana × G. triflora were treated by 50 μM oryzalin. This treatment produced diploid plants at various frequencies in all species ranging from 37.5% of G. triflora to 16.7% of G. triflora var. japonica f. nontana × G. scabra (Table 6). In addition to diploids, tetraploids and mixoploids were obtained in all species used. Optimum periods of treatment depended on species. In G. triflora, treatment for 2–4 weeks gave the highest production of diploid and tetraploid (82%), in contrast, in two other species, treatment for 1 week showed the highest frequency of diploid and tetraploid production.

Table 6 Effect of oryzalin treatment periods on chromosome doubling in gentians
Species
 (No. of genotype used)		 Treatment period (w) No. of plants examined No. of plants (%)
x 2x 4x x + 2x x + 4x 2x + 4x
G. triflora
 (5 genotypesa)		 0 24 24 (100) 0 0 0 0 0
1 46 13 (28.3) 11 (23.9) 20 (43.5) 0 0 2 (4.3)
2 24 3 (12.5) 9 (37.5) 9 (37.5) 2 (8.3) 0 1 (4.2)
4 28 4 (14.3) 9 (32.1) 14 (50.0) 0 0 1 (3.6)
G. scabra
 (17-260 line)		 0 25 25 (100) 0 0 0 0 0
1 42 26 (61.9) 8 (19.0) 6 (14.3) 1 (2.4) 0 1 (2.4)
2 29 20 (69.0) 5 (17.2) 2 (6.9) 2 (6.9) 0 0
4 29 24 (82.8) 1 (3.4) 2 (6.9) 0 1 (3.4) 1 (3.4)
G. triflora var. japonica f. montana × G. triflora
 (17-771 line)		 0 10 10 (100) 0 0 0 0 0
1 11 6 (54.5) 2 (18.2) 3 (27.3) 0 0 0
2 12 10 (83.3) 2 (16.7) 0 0 0 0
4 2 2 (100) 0 0 0 0 0
a  Five genotype consist of cv. Ashiro-no-Aki, cv. Maciry, 06-6, 05-03B and 05-05B.

Discussion

Cold pretreatment was found to be beneficial for production of ELSs from unfertilized ovule culture of gentians in the present study. These results are consistent with those of unfertilized ovule/ovary culture in Beta vulgaris (Gurel et al. 2000, Lux et al. 1990) and Triticum durum (Sibi et al. 2001). On the other hand, no positive influences of cold pretreatment on gynogenic response were reported in Cucrubita pepo (Metwally et al. 1998) and Guizotia abyssinica (Bhat and Murthy 2007). In G. triflora, Pathirana et al. (2011) also recommended cold pretreatment (4°C for 48 h) on anther and ovary culture, though they did not show detailed data. Our results indicate that cold pretreatment for 1–2 weeks is beneficial for effective induction of ELSs in gentians, and as mentioned in Introduction, such pretreatment could be useful for avoidance of the concentrated ovule culture work within a limited time.

The donor genotype is known to be one of the most important factors in various tissue culture systems. In unfertilized ovule culture, the genotypic effect has also been reported in several species (reviewed by Bohance 2009, Chen et al. 2011). The present study using 43 genotypes including G. triflora, G. scabra, G. triflora var. japonica f. montana and their interspecific hybrids showed that 40 genotypes produced ELSs and regenerated plantlets in spite of genotypic variations in their frequency. Our previous report which used four genotypes indicated that although genotypic variations are present, unfertilized ovule culture was affected less by genotypes compared with that of anther culture (Doi et al. 2011). The present study reinforced the effectiveness of the unfertilized ovule culture for production of haploids and DHs, and revealed that although there is the effect of genotype in the frequency of ELS production, unfertilized ovule culture could be utilized for production of haploids and DHs among a wide range of gentian genotypes. Such genotypic variations and applicability of a wide range of genotypes are reported in gynogenesis of several plants such as onion, sugar beet and carrot (Bohanec and Jakse 1999, Geoffriau et al. 1997, Gurel et al. 2000, Kiełkowska and Adamus 2010).

Reproducibility of the results obtained by tissue culture techniques is a fundamental requirement in plant breeding as well as basic studies. To test such reproducibility and reliability of our culture system, we carried out unfertilized ovule culture in two different labs, at Iwate Univ. and at IARC. Our results revealed that all eight genotypes used as common materials in these two labs demonstrated the production of ELSs in both labs as well. In addition, other genotypes, which were used in each lab, also produced ELSs. These results indicated that unfertilized ovule culture system is reproducible and stable for production of haploids and DHs in gentians. On the other hand, the frequency of ELS production at Iwate Univ. was higher than that at IARC. Such a higher frequency of production at Iwate Univ. might have been due to the application of cold pretreatment of materials and/or the involvement of more experienced researchers on unfertilized ovule culture.

A large number of regenerated plantlets, which were obtained in this study, consisted of haploids, diploids, polyploids and chimeric ones and the majority of these plants were haploids (57.8%) and diploids (34.6%). However, their frequencies were different between species. More than half of the regenerants (66.4%) were haploids in G. triflora, whereas more than half of the regenerants (59.8%) were diploids in G. scabra. Such results were consistent with those of previous reports (Doi et al. 2011). Although we did not investigate whether diploid plants are DHs or not, we are certain that the majority of diploid plants are DHs, because we have already reported that 96% of diploids plants obtained via gynogenesis were identified as DHs based on DNA markers (Doi et al. 2011). DH plants could be also obtained from haploids by chromosome doubling treatment using oryzalin. In G. triflora, Morgan et al. (2003) obtained a tetraploid plant from the diploid by treatment with oryzalin. Our results showed that oryzalin is also effective for production of DH and tetraploid plants with high frequency in varied gentian genotypes. Not only DH plants but also tetraploid plants are important for breeding new cultivars in gentian such as development of gigantic flowers and triploid hybrid cultivars.

The present study revealed that unfertilized ovule culture of gentians is a powerful tool for obtaining haploids and DHs because of its application to a wide range of genotypes and its reproducible and reliable nature. Not only breeding programs using regenerated plants obtained in this study, but also genetic and developmental studies of gynogenesis, are currently being carried out.

Acknowledgments

This work was supported in part by “Research and Development Projects for Application in Promoting New Policy of Agriculture, Forestry and Fisheries” from the Ministry of Agriculture, Forestry and Fisheries of Japan.

Literature Cited
 
© 2013 by JAPANESE SOCIETY OF BREEDING
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