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In vitro induction of the amphiploid in interspecific hybrid of blueberry (Vaccinium corymbosum × Vaccinium ashei) with colchicine treatment
Scientia Horticulturae 122 (2009) 375–379
Contents lists available at ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
In vitro induction of the amphiploid in interspecific hybrid of blueberry (Vaccinium corymbosum Vaccinium ashei) with colchicine treatment Chieko Miyashita a,*, Shunji Ishikawa b, Masahiro Mii c a
Specialty Products Development Division, Tokyo Metropolitan Agriculture and Forestry Research Center, 3-8-1 Fuzimi-cho, Tachikawa-shi, Tokyo 190-0013, Japan Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509, Japan c Graduate School of Horticulture, Chiba University, 648 Matsudo, Matsudo-shi, Chiba 271-8510, Japan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 October 2008 Received in revised form 26 May 2009 Accepted 27 May 2009
Amphiploids were produced from pentaploid hybrids between Vaccinium corymbosum (4x) and Vaccinium ashei (6x) by colchicine treatment. Seeds of each parental species and those obtained by the interspecific crossing were treated with colchicine at 0, 500, 1000 and 2000 mg/l for 7 days, and the ploidy level of the seedlings was determined with flow cytometry. Either amphiploid (10x) or ploidy chimera (5x + 10x) was obtained from interspecific hybrid seeds treated with all colchicine concentrations, while no chromosome-doubled plants were obtained from both parents. Cross direction in interspecific hybridization affected the results of colchicine treatment and amphiploids were obtained only when V. corymbosum was used as the seed parent. In this cross, 17–25% of the seedlings turned to be amphidiploids or ploidy chimeras by treating with 500 mg/l colchicines for 7 days. These results indicate that susceptibility to colchicine may increase in the interspecific hybrid compared with the parental species but only when V. corymbosum was used as female parent. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Blueberry breeding Vaccinium corymbosum Vaccinium ashei Interspecific hybrid Colchicine Chromosome doubling
1. Introduction The blueberry is the general term for the small shrubs which belong to the genus Vaccinium section Cyanococcus. Major cultivated species of blueberry are highbush blueberry (Vaccinium corymbosum, 2n = 4x = 48) and rabbiteye blueberry (Vaccinium ashei, 2n = 6x = 60). The two species differ greatly in various characteristics. V. corymbosum has early maturity and cold tolerance, whereas V. ashei has late maturity and warm area adaptability. To increase blueberry cultivation, it is necessary to produce new cultivars, which have high adaptability to various climates and market needs. Therefore, breeding through interspecific hybridization between these two species might be attractive. Although it is possible to produce interspecific hybrids in this cross combination, fertility of the hybrids is generally low because of their pentaploid nature (Vorsa et al., 1987). Consequently, it is difficult to utilize directly the interspecific hybrids as breeding material. However, it might be possible that the 5x interspecific hybrids are utilized as new materials for cross
* Corresponding author. Present address: Hachijo Branch, Tokyo Metropolitan Islands Area Research and Development Center of Agriculture, Forestry and Fisheries, 4341-11 Ohkago Hachijo-machi, Tokyo 100-1401, Japan. Tel.: +81 49 962 0042; fax: +81 49 962 2083. E-mail address: [email protected] (C. Miyashita). 0304-4238/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2009.05.028
breeding if they are chromosome-doubled to produce amphiploids, which usually restore the fertility to some extent. To produce chromosome-doubled plants, colchicine treatment has mainly been used as an efficient strategy for various plant species. In fruit trees, colchicine has successfully been applied to produce polyploids in various crops such as grape (Notsuka et al., 2000), citrus (Gmitter and Ling, 1991), and loquat (Yahata et al., 2004). In blueberries, several successful results of chromosome doubling have been reported with colchicine treatment. Moore et al. (1964) induced a 10x shoot from a pentaploid hybrid of V. ashei V. corymbosum with colchicine treatment to axillary buds. Flowers on the 10x shoot produced abundant pollen which had larger and more normal shape than those of pentaploid. Thereafter, Jelenkovic and Draper (1970) reported that the 10x blueberry shoot induced by Moore et al. (1964) could produce progenies at the low rate by selfing and by crossing with 2x, 4x and 6x species of Vaccinium. These results suggest the possibility that amphipentaploid hybrids between V. corymbosum and V. ashei will be utilized as breeding material for producing the novel hybrids of blueberry at 10x level or other ploidy levels through backcrossing with either parental species. Especially, octaploids which are expected to generate by crossing 10x interspecific hybrid with V. ashei (6x) and also hexaploids generated by crossing the 8x with V. corymbosum (4x) are expected to have fertility, which can be possibly used for producing varieties that have unique genomic constitutions.
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C. Miyashita et al. / Scientia Horticulturae 122 (2009) 375–379
To confirm the results on the chromosome doubling treatment, assessment of the ploidy level has been conducted by counting the chromosome number of the plants obtained after the treatment with colchicine. However, the two important blueberry species described above are polyploids with the basic chromosome number of 12, and it is quite difficult to determine the accurate chromosome number of hyperploids obtained by chromosome doubling treatment (Lyrene and Perry, 1982). To facilitate the determination of ploidy levels, Costich et al. (1993) showed the effectiveness of DNA flow cytometry in blueberries. In this study, we determined optimum conditions for in vitro colchicine treatment to induce polyploids from interspecific hybrid seeds in order to produce large populations of 10x plants with genetic diversity for use in blueberry breeding. Moreover, we determined ploidy levels of colchicine-treated seedlings by flow cytometry for quick and easy screening of chromosome-doubled plants. 2. Materials and methods 2.1. Plant materials Three cultivars of V. corymbosum (‘Earliblue’, ‘Weymouth’, and ‘Spartan’) and two cultivars of V. ashei (‘Baldwin’, and ‘Homebell’) were used in reciprocal interspecific crosses. The seeds obtained from the interspecific crossings and those from open pollination, which were assumed to derive from intraspecific crossing, were used in the experiment. 2.2. In vitro seed sowing and low temperature treatment Low temperature treatment at 5 8C for 2 months was effective to promote seed germination of blueberry in the result of preliminary experiment. Therefore, the seeds collected from fruits were sterilized with sodium hypochlorite solution of 1% available chlorine for 15 min, sowed on 8 g/l agarose-solidified medium without any additives in 9 cm plastic Petri dish, and incubated at 5 8C for 2 months. For each dish, 30–88 seeds were sowed, and the dishes were sealed with Parafilm. 2.3. Colchicine treatment After the low temperature treatment, the seeds were put on a set of 5 filter papers (7 cm in diameter) wetted with 7 ml of colchicine solution (0, 500, 1000 and 2000 mg/l), in 9 cm plastic Petri dish, which was sealed with Parafilm in order to prevent the desiccation. Seven or 14 days later, the seeds were transferred onto the germination medium which consisted of WPM (Lloyd and McCown, 1981) as the basal medium containing 20 g/l sucrose and 3 g/l gellan gum, adjusted to pH 4.5, and dispensed into 9 cm plastic dishes each containing 30 ml medium. For each dish, 30–88 seeds were sowed and the dishes were sealed with Parafilm. The colchicine treatment and the seed culture were performed at 22 8C under continuous light condition. The effect of colchicine concentration on chromosome doubling was evaluated using interspecific hybrid seeds of Earliblue Baldwin and open pollinated seeds of Earliblue and Baldwin at 0, 500, 1000 or 2000 mg/l for 7 days. For each treatment, 3 replicates with each 10–37 seeds were made. To clarify the relationship between direction of interspecific crossings and doubling efficiency, seeds obtained by reciprocal crossings of both Weymouth Baldwin and Spartan Homebell were treated with colchicine at 500 mg/l for 7 days. For each crossing, 20–80 seeds were treated.
2.4. Assessment of seed germination and ploidy level Frequency of seed germination was investigated 12 or 24 weeks after the colchicine treatment by scoring the seeds with emerged cotyledons as germinated. Germinating rates were subjected to analysis of variance after arcsin transformation, and the means were compared using the least significant difference test. Ploidy level of the seedlings obtained after the treatment was determined by flow cytometry. The youngest leaf of the shoot, which grew about 2 cm in height, was cut into small pieces in a 200 ml solution A of Plant High Resolution DNA kit type P (Partec) for isolating nuclei, and stained with 40 ,6-diamidino-2phenylindole (DAPI) solution. After incubating for a few minutes, 1 ml of DAPI staining solution [10 mM Tris–HCl, pH 7.5, containing 50 mM sodium citrate, 2 mM MgCl2, 1% (w/v) PVP K-30 (Wako Pure Chemicals Industry Ltd., Osaka, Japan), 0.1% (v/ v) Triton X-100, 2.5 mg/l DAPI (40 ,6-diamidino-2-phenylindole dihydrochloride)] was added to stain the nuclei. The solution mixture was filtered through a 45 mm nylon mesh to remove debris and subjected to flow cytometric analysis using a PA flow cytometer (Partec GmbH, Munster, Germany) equipped with a mercury lamp. 3. Results 3.1. Effect of colchicine concentration on seed germination and chromosome doubling On medium containing no colchicine, the frequency of normally germinated seeds in the interspecific hybrid seeds of V. corymbosum (Earliblue) V. ashei (Baldwin) was 16.7% after 12 weeks and the hybrid seeds showed poor germination ability compared with open pollinated seeds of both parents: 86.7% in Earliblue and 66.7% in Baldwin, respectively (Table 1). Whereas the rate of callus formation in the hybrid seeds was 30.0%, which was extremely high compared with 3% or less in both parents. Inhibitory effect of colchicine treatment for 7 days on seed germination was evident in the interspecific hybrid seeds, and the germination frequency of the hybrid seeds was 3–4% irrespective of the concentrations tested in the present study (500–2000 mg/l), which was considerably low as compared with 16.7% in control seeds without colchicine treatment. The germination rate of open pollinated seeds of V. corymbosum was slightly but significantly decreased by 500 and 1000 mg/l colchicine treatment. In contrast, no significant Table 1 Effect of colchicine concentration on germination of blueberry seeds. Colchicine concentration (mg/l)
Germinating rate (%)a Normal
Callusb
Total
V. corymbosum V. ashei (Earliblue Baldwin)
0 500 1000 2000
16.7 3.2 3.3 4.2
a b b b
30.0 14.1 12.2 12.6
a b b b
46.7 17.3 15.6 16.8
a b b b
V. corymbosum Earliblue
0 500 1000 2000
86.7 66.4 65.5 77.3
a b b ab
3.3 2.7 2.7 2.7
ns ns ns ns
90.0 69.1 68.2 80.0
a b b ab
V. ashei Baldwin
0 500 1000 2000
66.7 66.0 55.4 60.0
ns ns ns ns
0.0 0.0 1.5 3.3
ns ns ns ns
66.7 66.0 56.9 63.3
ns ns ns ns
Progeny
a Germination rate was investigated till 12 weeks of colchicine treatment. Data are means from 3 replicates. Values in each group of seeds in each column followed by the same letter are not significantly different at the 0.05 level. ns: No significant difference. b Seeds which germinated abnormally into calli.
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Fig. 1. Flow cytometric analysis on DNA contents of interspecific hybrid and amphiploid of blueberry and their leaf morphology. (a) V. corymbosum (4x). (b) V. ashei (6x). (c) V. corymbosum V. ashei (5x). (d) Amphiploid of V. corymbosum V. ashei (10x). (e) Ploidy chimera of V. corymbosum V. ashei (5x + 10x). Note the thicker round leaf of the amphiploid (d). The bar represents 1 cm.
decrease in the germination rate was recognized in V. ashei seeds by the colchicine treatment. Relative nuclear DNA content of the seedlings assessed by flow cytometry of V. ashei (6x) was approximately 1.5 times as large as V. corymbosum (4x) and the seedlings derived from interspecific crossing were intermediate of the parents. Therefore, it was possible to clearly distinguish chromosome-doubled plants from both parents and the interspecific hybrids (Fig. 1). As for the interspecific hybrid seedlings, amphiploid (10x) or ploidy chimera (5x + 10x) occurred in all the colchicine concentrations tested (Table 2, Fig. 1). The rate of chromosome-doubled plants, which include amphiploid and ploidy chimera, was 33–50% of the seedlings obtained and no large difference was recognized among the colchicine concentrations used. In contrast, neither
polyploid nor ploidy chimera was obtained from open pollinated seedlings of either parents at any colchicine concentrations tested by using 70–82 seedlings for each treatment of V. corymbosum and 31–35 seedlings for each treatment of V. ashei, respectively. 3.2. Relationships between reciprocal crossings and germination or doubling Reciprocal crossings were performed in the two cross combinations, Weymouth (V. corymbosum) Baldwin (V. ashei) and Spartan (V. corymbosum) Homebell (V. ashei), and 4 groups of the seeds obtained were treated with colchicine. Without colchicine treatment, normal germination rate after 24 weeks was low (less than 20%) when V. corymbosum cultivars were used
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Table 2 Effect of colchicine concentration on chromosome doubling of blueberry seedlings. Progeny
Colchicine concentration (mg/l)
Ploidy level 4x
V. corymbosum V. ashei (Earliblue Baldwin)
a b
0 500 1000 2000
Doubling rateb (%)
Number of seedlings
Number of seedlings analyzeda
5 3 4 4
5x
6x
5 2 2 2
10x
5x + 10x
1 2 2
0.0 33.3 50.0 50.0
Seedlings obtained after colchicine treatment were subjected to flow cytometric analysis. Doubling rate was shown as the sum of amphiploid (10x) and ploidy chimera (5x + 10x).
Table 3 Effect of reciprocal crossing on germination of blueberry seeds. Femalea Malea
Colchicine concentration (mg/l)
Number of seeds sowed
Germinating rate (%)b Normalc
Callus
Total
V. corymbosum (We) V. ashei (Ba)
0 500
30 74
10.0 5.4 (54.1)
3.3 0.0
13.3 5.4
V. ashei (Ba) V. corymbosum (We)
0 500
30 36
76.7 69.4 (90.6)
0.0 0.0
76.7 69.4
V. corymbosum (Sp) V. ashei (Ho)
0 500
30 80
20.0 15.0 (75.0)
10.0 26.3
30.0 41.3
V. ashei (Ho) V. corymbosum (Sp)
0 500
30 56
86.7 82.1 (94.8)
0.0 0.0
86.7 82.1
a b c
V. corymbosum cultivars: Weymouth (We) and Spartan (Sp). V. ashei cultivars: Baldwin (Ba) and Homebell (Ho). Germination rate was obtained after 24 weeks of colchicine treatment. Values in parentheses show relative percentages provided that the percentage in no treatment is 100.
as seed parents in each reciprocal crossing combination, whereas seeds obtained from the crosses when V. ashei cultivars were seed parents showed more than 77% germination rates (Table 3). In addition, callus conversion occurred only when V. corymbosum cultivars were seed parents. Influence of colchicine treatment on the germination of hybrid seeds was remarkable when V. corymbosum was the seed parent, and normal germination rate with 500 mg/l colchicine treatment showed 25–46% decrease compared to the control. However, almost no decrease in the germination rate was found when the seed parent was V. ashei. Results of flow cytometry analysis on the colchicine-treated seedlings revealed that the amphiploid or ploidy chimera could be obtained in each crossing combination only when V. corymbosum was the seed parent (Table 4). Although many seedlings were obtained with the opposite cross direction, amphiploid plant was not obtained at all. By summarizing the data of Tables 2 and 4, the frequency of partial or complete chromosome-doubled plants obtained after the treatment of the seeds of V. corymbosum V. ashei with 500 mg/l colchicine for 7 days was 23.1%. 3.3. Growth characteristics of amphiploids The amphiploids and the ploidy chimeras induced from the seeds of V. corymbosum V. ashei with colchicine treatment grew
favorably in the greenhouse after acclimatization and potting. All amphiploids and many chimeras showed common characteristics such as thicker rounder leaves, thicker branches and slower growth compared to the parents and the other interspecific hybrid plants (5x) obtained in the present study (Fig. 1). While remaining chimeric plants were almost morphologically equal to the pentaploid hybrids. These results may suggest that distribution of the tissues with chromosome-doubled cells (10x cells) in these periclinal chimeras differed between the former and the later type. 4. Discussion The results of the present study clearly showed that chromosome-doubled plants (10x) and the ploidy chimera (5x + 10x) could be produced at the high frequency from the interspecific hybrid seeds (V. corymbosum V. ashei) by treating with 500 mg/l colchicine for 7 days. On the contrary, polyploids could not be obtained from the open pollinated seeds of each parental species and the interspecific hybrid seeds of the opposite crossing (V. ashei V. corymbosum) with the same colchicine treatment conditions. Since polyploids were successfully obtained from the both parental species used in the present study, V. ashei by Lyrene and Perry (1982), and V. corymbosum by Goldy and Lyrene (1984) with colchicine treatment of axillary buds of in vitro shoot, it is
Table 4 Effect of reciprocal crossing on chromosome doubling of blueberry seedlings after treating with 500 mg/l colchicine for 7 days. Female Male
Number of seedlings analyzed
Number of seedlings
Doubling rate (%)
Ploidy level 5x V. V. V. V.
corymbosum (We) V. ashei (Ba) ashei (Ba) V. corymbosum (We) corymbosum (Sp) V. ashei (Ho) ashei (Ho) V. corymbosum (Sp)
4 23 6 44
3 23 5 44
10x
5x + 10x 1
1
25.0 0.0 16.7 0.0
C. Miyashita et al. / Scientia Horticulturae 122 (2009) 375–379
possible that apical meristem of embryo and that of axillary bud of shoot are different in sensitivity to colchicine. Another possible reason for the successful chromosome doubling in V. corymbosum V. ashei might be that interspecific hybrid genome is easier to be chromosome-doubled than the both parent genomes. Perry and Lyrene (1984) reported that spontaneous polyploids were obtained more frequently in the interspecific hybrid than the both parents in the study of colchicine treatment on in vitro shoot cuttings of V. darrowi, V. elliottii and their interspecific hybrid. Therefore, interspecific hybrid genomes in Vaccinium species might have some instability, which might be stabilized in the amphidiploid. However, such a tendency was not recognized in the seeds of opposite cross (V. ashei V. corymbosum) in the present study. Although the difference in sensitivity to colchicine in the reciprocal crosses is therefore still not clear but it might be necessary to consider the effect of the cytoplasmic genome in the response to colchicine in such interspecific hybridization. In interspecific hybrid seeds (V. corymbosum V. ashei), normal germination rate was low (20% or less) for all the cross combinations, whereas frequency of abnormal germination such as callus formation was high. However, callus conversion rate differed greatly in the range of 3–30% depending on the cross combination of the cultivars. These results suggest that germination ability of the interspecific hybrid seeds differs largely depending upon genotype of the parents, and that in vitro seed culture is effective for promoting germination and growth of interspecific hybrid seeds, whose germination ability ex vitro is low. To obtain hybrid plants in this interspecific cross, it is important to prevent callusing of the seeds or to establish the method for plant regeneration from calli, which might also be important for in vitro amphiploid production from the hybrids. In the present study, ploidy level (5x, 10x or 5x + 10x) of blueberry seedlings was clearly identified by flow cytometry. In addition, a large number of seedlings could be analyzed without spending much time since the time required for analysis per plant was a few minutes. In order to screen polyploids of V. ashei, Lyrene and Perry (1982) selected the shoots with unusually large diameter after in vitro colchicine treatment and confirmed all the selected shoots as polyploids by counting the chromosome number. However, it is rather difficult to select the shoots with thicker stem at the early stage after colchicine treatment and the ploidy chimeras induced in the present study also had shoots of large diameter. In the case of ploidy chimera, there is a great risk that it reverts into original ploidy level during the subsequent growth. Therefore, flow cytometry is more useful not only to accurately evaluate the effect of colchicine on chromosome doubling but also to rapidly identify the objective true polyploids at the early stage after the treatment. The method of chromosome doubling by in vitro colchicine treatment to seeds and the determination of ploidy level of the seedlings after the treatment by flow cytometry used in this study are effective for obtaining the amphiploids of the V.
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corymbosum V. ashei interspecific hybrid. Both the amphiploids (10x) and ploidy chimeras of the hybrids induced in the present study are growing normally without any serious growth depressions. Although the amphiploids are usually expected to have fertility, it is necessary to perform detailed studies on pollen fertility and chromosome pairing at meiosis for their subsequent utilization as breeding materials since high ploidy levels sometimes cause low fertility due to abnormality in meiosis. In addition, it is also important to evaluate the pentaploid interspecific hybrids produced in the present study for their own use as bridge materials to transfer genes between the parent species because the same type of interspecific hybrids (5x) produced by the previous studies had a certain level of fertility and the progenies obtained by backcrossing with either parent had higher fertility (Vorsa et al., 1987; Laverty and Vorsa, 1991). Moreover, it should also be necessary to evaluate the ploidy chimeras obtained in the present study, since periclinal ploidy chimera in Meiwa kumquat (Fortunella crassifolia) has valuable characteristics created by chimeric nature such as larger fruit size than those of the tetraploid line, and fewer seeds and thicker pericarp than the diploid line (Yasuda et al., 2008). Stability of the chimera and characterization of various characters including the fruit quality of these ploidy chimera plants will be important for their further use in the cultivation of this fruit crop. References Costich, D.E., Ortiz, R., Meagher, T.R., Bruederle, L.P., Vorsa, N., 1993. Determination of ploidy level and nuclear DNA content in blueberry by flow cytometry. Theor. Appl. Genet. 86, 1001–1006. Gmitter Jr., F.G., Ling, K., 1991. Embryogenesis in vitro and nonchimeric tetraploid plant recovery from undeveloped citrus ovules treated with colchicine. J. Am. Soc. Hort. Sci. 116, 317–321. Goldy, R.G., Lyrene, P.M., 1984. In vitro colchicine treatment of 4x blueberries, Vaccinium sp. J. Am. Soc. Hort. Sci. 109, 336–338. Jelenkovic, G., Draper, D., 1970. Fertility and chromosome behavior of a derived decaploid of Vaccinium. J. Am. Soc. Hort. Sci. 95, 816–820. Laverty, T., Vorsa, N., 1991. Fertility of aneuploids between the 5x and 6x levels in blueberry: the potential for gene transfer from 4x to 6x levels. J. Am. Soc. Hort. Sci. 116, 330–335. Lloyd, G., McCown, B.H., 1981. Commercially-feasible micropropagation of Mountain Laurel, Kalmia latifolia, by shoot tip culture. Proc. Int. Plant Prop. Soc. 30, 421–427. Lyrene, P.M., Perry, J.L., 1982. Production and selection of blueberry polyploids in vitro. J. Hered. 73, 377–378. Moore, J.N., Scott, D.H., Dermen, H., 1964. Development of a decaploid blueberry by colchicine treatment. Proc. Am. Soc. Hort. Sci. 84, 274–279. Notsuka, K., Tsuru, T., Shiraishi, M., 2000. Induced polyploid grapes via in vitro chromosome doubling. J. Jpn. Soc. Hort. Sci. 69, 543–551. Perry, J.L., Lyrene, P.M., 1984. In vitro induction of tetraploidy in Vaccinium darrowi, V. elliottii, and V. darrowi V. elliottii with colchicine treatment. J. Am. Soc. Hort. Sci. 109, 4–6. Vorsa, N., Jelenkovic, G., Draper, A.D., Welker, W.V., 1987. Fertility of 4x 5 and 5x 4x progenies derived from Vaccinium ashei/corymbosum pentaploid hybrids. J. Am. Soc. Hort. Sci. 112, 993–997. Yahata, S., Sato, S., Ohara, H., Matsui, H., 2004. Induction of tetraploid in loquat with amiprofos-methyl and colchicine. Hort. Res. (Japan) 3, 339–344. Yasuda, K., Kunitake, H., Nakagawa, S., Kurogi, H., Yahata, M., Hirata, R., Yoshikura, Y., Kawakami, I., Sugimoto, Y., 2008. The confirmation of ploidy periclinal chimera and its morphological characteristics in Meiwa kumquat ‘Yubeni’. Hort. Res. (Japan) 7, 165–171.
Contents lists available at ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
In vitro induction of the amphiploid in interspecific hybrid of blueberry (Vaccinium corymbosum Vaccinium ashei) with colchicine treatment Chieko Miyashita a,*, Shunji Ishikawa b, Masahiro Mii c a
Specialty Products Development Division, Tokyo Metropolitan Agriculture and Forestry Research Center, 3-8-1 Fuzimi-cho, Tachikawa-shi, Tokyo 190-0013, Japan Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509, Japan c Graduate School of Horticulture, Chiba University, 648 Matsudo, Matsudo-shi, Chiba 271-8510, Japan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 October 2008 Received in revised form 26 May 2009 Accepted 27 May 2009
Amphiploids were produced from pentaploid hybrids between Vaccinium corymbosum (4x) and Vaccinium ashei (6x) by colchicine treatment. Seeds of each parental species and those obtained by the interspecific crossing were treated with colchicine at 0, 500, 1000 and 2000 mg/l for 7 days, and the ploidy level of the seedlings was determined with flow cytometry. Either amphiploid (10x) or ploidy chimera (5x + 10x) was obtained from interspecific hybrid seeds treated with all colchicine concentrations, while no chromosome-doubled plants were obtained from both parents. Cross direction in interspecific hybridization affected the results of colchicine treatment and amphiploids were obtained only when V. corymbosum was used as the seed parent. In this cross, 17–25% of the seedlings turned to be amphidiploids or ploidy chimeras by treating with 500 mg/l colchicines for 7 days. These results indicate that susceptibility to colchicine may increase in the interspecific hybrid compared with the parental species but only when V. corymbosum was used as female parent. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Blueberry breeding Vaccinium corymbosum Vaccinium ashei Interspecific hybrid Colchicine Chromosome doubling
1. Introduction The blueberry is the general term for the small shrubs which belong to the genus Vaccinium section Cyanococcus. Major cultivated species of blueberry are highbush blueberry (Vaccinium corymbosum, 2n = 4x = 48) and rabbiteye blueberry (Vaccinium ashei, 2n = 6x = 60). The two species differ greatly in various characteristics. V. corymbosum has early maturity and cold tolerance, whereas V. ashei has late maturity and warm area adaptability. To increase blueberry cultivation, it is necessary to produce new cultivars, which have high adaptability to various climates and market needs. Therefore, breeding through interspecific hybridization between these two species might be attractive. Although it is possible to produce interspecific hybrids in this cross combination, fertility of the hybrids is generally low because of their pentaploid nature (Vorsa et al., 1987). Consequently, it is difficult to utilize directly the interspecific hybrids as breeding material. However, it might be possible that the 5x interspecific hybrids are utilized as new materials for cross
* Corresponding author. Present address: Hachijo Branch, Tokyo Metropolitan Islands Area Research and Development Center of Agriculture, Forestry and Fisheries, 4341-11 Ohkago Hachijo-machi, Tokyo 100-1401, Japan. Tel.: +81 49 962 0042; fax: +81 49 962 2083. E-mail address: [email protected] (C. Miyashita). 0304-4238/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2009.05.028
breeding if they are chromosome-doubled to produce amphiploids, which usually restore the fertility to some extent. To produce chromosome-doubled plants, colchicine treatment has mainly been used as an efficient strategy for various plant species. In fruit trees, colchicine has successfully been applied to produce polyploids in various crops such as grape (Notsuka et al., 2000), citrus (Gmitter and Ling, 1991), and loquat (Yahata et al., 2004). In blueberries, several successful results of chromosome doubling have been reported with colchicine treatment. Moore et al. (1964) induced a 10x shoot from a pentaploid hybrid of V. ashei V. corymbosum with colchicine treatment to axillary buds. Flowers on the 10x shoot produced abundant pollen which had larger and more normal shape than those of pentaploid. Thereafter, Jelenkovic and Draper (1970) reported that the 10x blueberry shoot induced by Moore et al. (1964) could produce progenies at the low rate by selfing and by crossing with 2x, 4x and 6x species of Vaccinium. These results suggest the possibility that amphipentaploid hybrids between V. corymbosum and V. ashei will be utilized as breeding material for producing the novel hybrids of blueberry at 10x level or other ploidy levels through backcrossing with either parental species. Especially, octaploids which are expected to generate by crossing 10x interspecific hybrid with V. ashei (6x) and also hexaploids generated by crossing the 8x with V. corymbosum (4x) are expected to have fertility, which can be possibly used for producing varieties that have unique genomic constitutions.
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To confirm the results on the chromosome doubling treatment, assessment of the ploidy level has been conducted by counting the chromosome number of the plants obtained after the treatment with colchicine. However, the two important blueberry species described above are polyploids with the basic chromosome number of 12, and it is quite difficult to determine the accurate chromosome number of hyperploids obtained by chromosome doubling treatment (Lyrene and Perry, 1982). To facilitate the determination of ploidy levels, Costich et al. (1993) showed the effectiveness of DNA flow cytometry in blueberries. In this study, we determined optimum conditions for in vitro colchicine treatment to induce polyploids from interspecific hybrid seeds in order to produce large populations of 10x plants with genetic diversity for use in blueberry breeding. Moreover, we determined ploidy levels of colchicine-treated seedlings by flow cytometry for quick and easy screening of chromosome-doubled plants. 2. Materials and methods 2.1. Plant materials Three cultivars of V. corymbosum (‘Earliblue’, ‘Weymouth’, and ‘Spartan’) and two cultivars of V. ashei (‘Baldwin’, and ‘Homebell’) were used in reciprocal interspecific crosses. The seeds obtained from the interspecific crossings and those from open pollination, which were assumed to derive from intraspecific crossing, were used in the experiment. 2.2. In vitro seed sowing and low temperature treatment Low temperature treatment at 5 8C for 2 months was effective to promote seed germination of blueberry in the result of preliminary experiment. Therefore, the seeds collected from fruits were sterilized with sodium hypochlorite solution of 1% available chlorine for 15 min, sowed on 8 g/l agarose-solidified medium without any additives in 9 cm plastic Petri dish, and incubated at 5 8C for 2 months. For each dish, 30–88 seeds were sowed, and the dishes were sealed with Parafilm. 2.3. Colchicine treatment After the low temperature treatment, the seeds were put on a set of 5 filter papers (7 cm in diameter) wetted with 7 ml of colchicine solution (0, 500, 1000 and 2000 mg/l), in 9 cm plastic Petri dish, which was sealed with Parafilm in order to prevent the desiccation. Seven or 14 days later, the seeds were transferred onto the germination medium which consisted of WPM (Lloyd and McCown, 1981) as the basal medium containing 20 g/l sucrose and 3 g/l gellan gum, adjusted to pH 4.5, and dispensed into 9 cm plastic dishes each containing 30 ml medium. For each dish, 30–88 seeds were sowed and the dishes were sealed with Parafilm. The colchicine treatment and the seed culture were performed at 22 8C under continuous light condition. The effect of colchicine concentration on chromosome doubling was evaluated using interspecific hybrid seeds of Earliblue Baldwin and open pollinated seeds of Earliblue and Baldwin at 0, 500, 1000 or 2000 mg/l for 7 days. For each treatment, 3 replicates with each 10–37 seeds were made. To clarify the relationship between direction of interspecific crossings and doubling efficiency, seeds obtained by reciprocal crossings of both Weymouth Baldwin and Spartan Homebell were treated with colchicine at 500 mg/l for 7 days. For each crossing, 20–80 seeds were treated.
2.4. Assessment of seed germination and ploidy level Frequency of seed germination was investigated 12 or 24 weeks after the colchicine treatment by scoring the seeds with emerged cotyledons as germinated. Germinating rates were subjected to analysis of variance after arcsin transformation, and the means were compared using the least significant difference test. Ploidy level of the seedlings obtained after the treatment was determined by flow cytometry. The youngest leaf of the shoot, which grew about 2 cm in height, was cut into small pieces in a 200 ml solution A of Plant High Resolution DNA kit type P (Partec) for isolating nuclei, and stained with 40 ,6-diamidino-2phenylindole (DAPI) solution. After incubating for a few minutes, 1 ml of DAPI staining solution [10 mM Tris–HCl, pH 7.5, containing 50 mM sodium citrate, 2 mM MgCl2, 1% (w/v) PVP K-30 (Wako Pure Chemicals Industry Ltd., Osaka, Japan), 0.1% (v/ v) Triton X-100, 2.5 mg/l DAPI (40 ,6-diamidino-2-phenylindole dihydrochloride)] was added to stain the nuclei. The solution mixture was filtered through a 45 mm nylon mesh to remove debris and subjected to flow cytometric analysis using a PA flow cytometer (Partec GmbH, Munster, Germany) equipped with a mercury lamp. 3. Results 3.1. Effect of colchicine concentration on seed germination and chromosome doubling On medium containing no colchicine, the frequency of normally germinated seeds in the interspecific hybrid seeds of V. corymbosum (Earliblue) V. ashei (Baldwin) was 16.7% after 12 weeks and the hybrid seeds showed poor germination ability compared with open pollinated seeds of both parents: 86.7% in Earliblue and 66.7% in Baldwin, respectively (Table 1). Whereas the rate of callus formation in the hybrid seeds was 30.0%, which was extremely high compared with 3% or less in both parents. Inhibitory effect of colchicine treatment for 7 days on seed germination was evident in the interspecific hybrid seeds, and the germination frequency of the hybrid seeds was 3–4% irrespective of the concentrations tested in the present study (500–2000 mg/l), which was considerably low as compared with 16.7% in control seeds without colchicine treatment. The germination rate of open pollinated seeds of V. corymbosum was slightly but significantly decreased by 500 and 1000 mg/l colchicine treatment. In contrast, no significant Table 1 Effect of colchicine concentration on germination of blueberry seeds. Colchicine concentration (mg/l)
Germinating rate (%)a Normal
Callusb
Total
V. corymbosum V. ashei (Earliblue Baldwin)
0 500 1000 2000
16.7 3.2 3.3 4.2
a b b b
30.0 14.1 12.2 12.6
a b b b
46.7 17.3 15.6 16.8
a b b b
V. corymbosum Earliblue
0 500 1000 2000
86.7 66.4 65.5 77.3
a b b ab
3.3 2.7 2.7 2.7
ns ns ns ns
90.0 69.1 68.2 80.0
a b b ab
V. ashei Baldwin
0 500 1000 2000
66.7 66.0 55.4 60.0
ns ns ns ns
0.0 0.0 1.5 3.3
ns ns ns ns
66.7 66.0 56.9 63.3
ns ns ns ns
Progeny
a Germination rate was investigated till 12 weeks of colchicine treatment. Data are means from 3 replicates. Values in each group of seeds in each column followed by the same letter are not significantly different at the 0.05 level. ns: No significant difference. b Seeds which germinated abnormally into calli.
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Fig. 1. Flow cytometric analysis on DNA contents of interspecific hybrid and amphiploid of blueberry and their leaf morphology. (a) V. corymbosum (4x). (b) V. ashei (6x). (c) V. corymbosum V. ashei (5x). (d) Amphiploid of V. corymbosum V. ashei (10x). (e) Ploidy chimera of V. corymbosum V. ashei (5x + 10x). Note the thicker round leaf of the amphiploid (d). The bar represents 1 cm.
decrease in the germination rate was recognized in V. ashei seeds by the colchicine treatment. Relative nuclear DNA content of the seedlings assessed by flow cytometry of V. ashei (6x) was approximately 1.5 times as large as V. corymbosum (4x) and the seedlings derived from interspecific crossing were intermediate of the parents. Therefore, it was possible to clearly distinguish chromosome-doubled plants from both parents and the interspecific hybrids (Fig. 1). As for the interspecific hybrid seedlings, amphiploid (10x) or ploidy chimera (5x + 10x) occurred in all the colchicine concentrations tested (Table 2, Fig. 1). The rate of chromosome-doubled plants, which include amphiploid and ploidy chimera, was 33–50% of the seedlings obtained and no large difference was recognized among the colchicine concentrations used. In contrast, neither
polyploid nor ploidy chimera was obtained from open pollinated seedlings of either parents at any colchicine concentrations tested by using 70–82 seedlings for each treatment of V. corymbosum and 31–35 seedlings for each treatment of V. ashei, respectively. 3.2. Relationships between reciprocal crossings and germination or doubling Reciprocal crossings were performed in the two cross combinations, Weymouth (V. corymbosum) Baldwin (V. ashei) and Spartan (V. corymbosum) Homebell (V. ashei), and 4 groups of the seeds obtained were treated with colchicine. Without colchicine treatment, normal germination rate after 24 weeks was low (less than 20%) when V. corymbosum cultivars were used
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Table 2 Effect of colchicine concentration on chromosome doubling of blueberry seedlings. Progeny
Colchicine concentration (mg/l)
Ploidy level 4x
V. corymbosum V. ashei (Earliblue Baldwin)
a b
0 500 1000 2000
Doubling rateb (%)
Number of seedlings
Number of seedlings analyzeda
5 3 4 4
5x
6x
5 2 2 2
10x
5x + 10x
1 2 2
0.0 33.3 50.0 50.0
Seedlings obtained after colchicine treatment were subjected to flow cytometric analysis. Doubling rate was shown as the sum of amphiploid (10x) and ploidy chimera (5x + 10x).
Table 3 Effect of reciprocal crossing on germination of blueberry seeds. Femalea Malea
Colchicine concentration (mg/l)
Number of seeds sowed
Germinating rate (%)b Normalc
Callus
Total
V. corymbosum (We) V. ashei (Ba)
0 500
30 74
10.0 5.4 (54.1)
3.3 0.0
13.3 5.4
V. ashei (Ba) V. corymbosum (We)
0 500
30 36
76.7 69.4 (90.6)
0.0 0.0
76.7 69.4
V. corymbosum (Sp) V. ashei (Ho)
0 500
30 80
20.0 15.0 (75.0)
10.0 26.3
30.0 41.3
V. ashei (Ho) V. corymbosum (Sp)
0 500
30 56
86.7 82.1 (94.8)
0.0 0.0
86.7 82.1
a b c
V. corymbosum cultivars: Weymouth (We) and Spartan (Sp). V. ashei cultivars: Baldwin (Ba) and Homebell (Ho). Germination rate was obtained after 24 weeks of colchicine treatment. Values in parentheses show relative percentages provided that the percentage in no treatment is 100.
as seed parents in each reciprocal crossing combination, whereas seeds obtained from the crosses when V. ashei cultivars were seed parents showed more than 77% germination rates (Table 3). In addition, callus conversion occurred only when V. corymbosum cultivars were seed parents. Influence of colchicine treatment on the germination of hybrid seeds was remarkable when V. corymbosum was the seed parent, and normal germination rate with 500 mg/l colchicine treatment showed 25–46% decrease compared to the control. However, almost no decrease in the germination rate was found when the seed parent was V. ashei. Results of flow cytometry analysis on the colchicine-treated seedlings revealed that the amphiploid or ploidy chimera could be obtained in each crossing combination only when V. corymbosum was the seed parent (Table 4). Although many seedlings were obtained with the opposite cross direction, amphiploid plant was not obtained at all. By summarizing the data of Tables 2 and 4, the frequency of partial or complete chromosome-doubled plants obtained after the treatment of the seeds of V. corymbosum V. ashei with 500 mg/l colchicine for 7 days was 23.1%. 3.3. Growth characteristics of amphiploids The amphiploids and the ploidy chimeras induced from the seeds of V. corymbosum V. ashei with colchicine treatment grew
favorably in the greenhouse after acclimatization and potting. All amphiploids and many chimeras showed common characteristics such as thicker rounder leaves, thicker branches and slower growth compared to the parents and the other interspecific hybrid plants (5x) obtained in the present study (Fig. 1). While remaining chimeric plants were almost morphologically equal to the pentaploid hybrids. These results may suggest that distribution of the tissues with chromosome-doubled cells (10x cells) in these periclinal chimeras differed between the former and the later type. 4. Discussion The results of the present study clearly showed that chromosome-doubled plants (10x) and the ploidy chimera (5x + 10x) could be produced at the high frequency from the interspecific hybrid seeds (V. corymbosum V. ashei) by treating with 500 mg/l colchicine for 7 days. On the contrary, polyploids could not be obtained from the open pollinated seeds of each parental species and the interspecific hybrid seeds of the opposite crossing (V. ashei V. corymbosum) with the same colchicine treatment conditions. Since polyploids were successfully obtained from the both parental species used in the present study, V. ashei by Lyrene and Perry (1982), and V. corymbosum by Goldy and Lyrene (1984) with colchicine treatment of axillary buds of in vitro shoot, it is
Table 4 Effect of reciprocal crossing on chromosome doubling of blueberry seedlings after treating with 500 mg/l colchicine for 7 days. Female Male
Number of seedlings analyzed
Number of seedlings
Doubling rate (%)
Ploidy level 5x V. V. V. V.
corymbosum (We) V. ashei (Ba) ashei (Ba) V. corymbosum (We) corymbosum (Sp) V. ashei (Ho) ashei (Ho) V. corymbosum (Sp)
4 23 6 44
3 23 5 44
10x
5x + 10x 1
1
25.0 0.0 16.7 0.0
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possible that apical meristem of embryo and that of axillary bud of shoot are different in sensitivity to colchicine. Another possible reason for the successful chromosome doubling in V. corymbosum V. ashei might be that interspecific hybrid genome is easier to be chromosome-doubled than the both parent genomes. Perry and Lyrene (1984) reported that spontaneous polyploids were obtained more frequently in the interspecific hybrid than the both parents in the study of colchicine treatment on in vitro shoot cuttings of V. darrowi, V. elliottii and their interspecific hybrid. Therefore, interspecific hybrid genomes in Vaccinium species might have some instability, which might be stabilized in the amphidiploid. However, such a tendency was not recognized in the seeds of opposite cross (V. ashei V. corymbosum) in the present study. Although the difference in sensitivity to colchicine in the reciprocal crosses is therefore still not clear but it might be necessary to consider the effect of the cytoplasmic genome in the response to colchicine in such interspecific hybridization. In interspecific hybrid seeds (V. corymbosum V. ashei), normal germination rate was low (20% or less) for all the cross combinations, whereas frequency of abnormal germination such as callus formation was high. However, callus conversion rate differed greatly in the range of 3–30% depending on the cross combination of the cultivars. These results suggest that germination ability of the interspecific hybrid seeds differs largely depending upon genotype of the parents, and that in vitro seed culture is effective for promoting germination and growth of interspecific hybrid seeds, whose germination ability ex vitro is low. To obtain hybrid plants in this interspecific cross, it is important to prevent callusing of the seeds or to establish the method for plant regeneration from calli, which might also be important for in vitro amphiploid production from the hybrids. In the present study, ploidy level (5x, 10x or 5x + 10x) of blueberry seedlings was clearly identified by flow cytometry. In addition, a large number of seedlings could be analyzed without spending much time since the time required for analysis per plant was a few minutes. In order to screen polyploids of V. ashei, Lyrene and Perry (1982) selected the shoots with unusually large diameter after in vitro colchicine treatment and confirmed all the selected shoots as polyploids by counting the chromosome number. However, it is rather difficult to select the shoots with thicker stem at the early stage after colchicine treatment and the ploidy chimeras induced in the present study also had shoots of large diameter. In the case of ploidy chimera, there is a great risk that it reverts into original ploidy level during the subsequent growth. Therefore, flow cytometry is more useful not only to accurately evaluate the effect of colchicine on chromosome doubling but also to rapidly identify the objective true polyploids at the early stage after the treatment. The method of chromosome doubling by in vitro colchicine treatment to seeds and the determination of ploidy level of the seedlings after the treatment by flow cytometry used in this study are effective for obtaining the amphiploids of the V.
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corymbosum V. ashei interspecific hybrid. Both the amphiploids (10x) and ploidy chimeras of the hybrids induced in the present study are growing normally without any serious growth depressions. Although the amphiploids are usually expected to have fertility, it is necessary to perform detailed studies on pollen fertility and chromosome pairing at meiosis for their subsequent utilization as breeding materials since high ploidy levels sometimes cause low fertility due to abnormality in meiosis. In addition, it is also important to evaluate the pentaploid interspecific hybrids produced in the present study for their own use as bridge materials to transfer genes between the parent species because the same type of interspecific hybrids (5x) produced by the previous studies had a certain level of fertility and the progenies obtained by backcrossing with either parent had higher fertility (Vorsa et al., 1987; Laverty and Vorsa, 1991). Moreover, it should also be necessary to evaluate the ploidy chimeras obtained in the present study, since periclinal ploidy chimera in Meiwa kumquat (Fortunella crassifolia) has valuable characteristics created by chimeric nature such as larger fruit size than those of the tetraploid line, and fewer seeds and thicker pericarp than the diploid line (Yasuda et al., 2008). Stability of the chimera and characterization of various characters including the fruit quality of these ploidy chimera plants will be important for their further use in the cultivation of this fruit crop. References Costich, D.E., Ortiz, R., Meagher, T.R., Bruederle, L.P., Vorsa, N., 1993. Determination of ploidy level and nuclear DNA content in blueberry by flow cytometry. Theor. Appl. Genet. 86, 1001–1006. Gmitter Jr., F.G., Ling, K., 1991. Embryogenesis in vitro and nonchimeric tetraploid plant recovery from undeveloped citrus ovules treated with colchicine. J. Am. Soc. Hort. Sci. 116, 317–321. Goldy, R.G., Lyrene, P.M., 1984. In vitro colchicine treatment of 4x blueberries, Vaccinium sp. J. Am. Soc. Hort. Sci. 109, 336–338. Jelenkovic, G., Draper, D., 1970. Fertility and chromosome behavior of a derived decaploid of Vaccinium. J. Am. Soc. Hort. Sci. 95, 816–820. Laverty, T., Vorsa, N., 1991. Fertility of aneuploids between the 5x and 6x levels in blueberry: the potential for gene transfer from 4x to 6x levels. J. Am. Soc. Hort. Sci. 116, 330–335. Lloyd, G., McCown, B.H., 1981. Commercially-feasible micropropagation of Mountain Laurel, Kalmia latifolia, by shoot tip culture. Proc. Int. Plant Prop. Soc. 30, 421–427. Lyrene, P.M., Perry, J.L., 1982. Production and selection of blueberry polyploids in vitro. J. Hered. 73, 377–378. Moore, J.N., Scott, D.H., Dermen, H., 1964. Development of a decaploid blueberry by colchicine treatment. Proc. Am. Soc. Hort. Sci. 84, 274–279. Notsuka, K., Tsuru, T., Shiraishi, M., 2000. Induced polyploid grapes via in vitro chromosome doubling. J. Jpn. Soc. Hort. Sci. 69, 543–551. Perry, J.L., Lyrene, P.M., 1984. In vitro induction of tetraploidy in Vaccinium darrowi, V. elliottii, and V. darrowi V. elliottii with colchicine treatment. J. Am. Soc. Hort. Sci. 109, 4–6. Vorsa, N., Jelenkovic, G., Draper, A.D., Welker, W.V., 1987. Fertility of 4x 5 and 5x 4x progenies derived from Vaccinium ashei/corymbosum pentaploid hybrids. J. Am. Soc. Hort. Sci. 112, 993–997. Yahata, S., Sato, S., Ohara, H., Matsui, H., 2004. Induction of tetraploid in loquat with amiprofos-methyl and colchicine. Hort. Res. (Japan) 3, 339–344. Yasuda, K., Kunitake, H., Nakagawa, S., Kurogi, H., Yahata, M., Hirata, R., Yoshikura, Y., Kawakami, I., Sugimoto, Y., 2008. The confirmation of ploidy periclinal chimera and its morphological characteristics in Meiwa kumquat ‘Yubeni’. Hort. Res. (Japan) 7, 165–171.