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The Horticulture Journal 84 (1): 30–36. 2015. e Japanese Society for doi: 10.2503/hortj.MI-005 JSHS Horticultural Science http://www.jshs.jp/
Morphological Characteristics of a Doubled Haploid Line from ‘Banpeiyu’ Pummelo [Citrus maxima (Burm.) Merr.] and Its Reproductive Function
Masaki Yahata1, Tsunaki Nukaya1, Miki Sudo1, Tomohiro Ohta1, Kiichi Yasuda2, Hidehiro Inagaki1, Hiroo Mukai1, Hisashi Harada1, Toshihiko Takagi1, Haruki Komatsu3 and Hisato Kunitake2*
1Graduate School of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan 2Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan 3School of Agriculture, Tokai University, Kumamoto 869-1404, Japan
To obtain basic information about doubled haploid plants in Citrus, in the present study, we investigated the morphological characteristics in doubled haploid induced by colchicine-treated axillary shoot buds of a haploid plant from ‘Banpeiyu’ pummelo [C. maxima (Burm.) Merr.]. We also evaluated the reproductive potential of the doubled haploid as a male or a female parent by crossing with some diploids. In term of the results, this doubled haploid had significantly large leaf, flower and fruit compared with those of the original haploid plant. Moreover, the doubled haploid showed higher pollen fertility (84.1% stainability and 32.9% pollen germination rate) and a larger number of seeds (47.2 developed seeds per open-pollinated fruit) than the haploid. In the reciprocal crosses between the doubled haploid and some diploids, many developed seeds were obtained. These seeds germinated normally and developed into diploid seedlings. These results show that the doubled haploid will be valuable for genetic analysis and possibly for planned breeding.
Key Words: chromosome, homozygosity, pollen fertility, reciprocal cross.
1996; Hesse, 1971; Höfer and Grafe, 2003; Ochatt and Introduction Zhang, 1996; Zhang and Lespinasse, 1991) and sponta- Haploid and doubled haploid plants are of great value neous chromosome doubling of androgenetic micro- for genetic analysis and premeditated breeding (Bajaj, spores in anther culture (Höfer et al., 2008; Okada 1990; Germanà, 2006; Ochatt and Zhang, 1996). This is et al., 2009; Vanwynsberghe et al., 2005). especially the case for woody species, which are gener- In Citrus and related genera, some haploid plants ally characterized by a long reproductive cycle, a high have been produced by various techniques such as an- degree of heterozygosity, a large plant size, and self- ther culture (Germanà and Chiancone, 2003; Germanà incompatibility. et al., 1994; Hidaka et al., 1979), interploid hybridiza- In fruit crops, therefore, haploids have been obtained tion (Germanà and Chiancone, 2001; Oiyama and from some species such as kiwifruit [Actinidia deliciosa Kobayashi, 1993; Toolapong et al., 1996), and pollina- (A. Chev.) C. F. Liang & A. R. Ferguson], apple (Malus tion of irradiated pollen (Aleza et al., 2009; Froelicher pumila Mill.), banana (Musa acuminata Colla), sweet et al., 2007; Yahata et al., 2010). However, doubled cherry [Prunus avium (L.) L.], peach [P. persica (L.) haploid plants have only been induced by anther culture Batsch], and Japanese pear [Pyrus pyrifolia (Burm. f.) in Clementine mandarin (C. clementina hort. ex Nakai], and doubled haploids have also been induced Tanaka) (Germanà and Chiancone, 2003) and by treating shoots and leaves of the haploids with anti- ‘Valencia’ sweet orange [C. sinensis (L.) Osbeck] (Cao mitotic agents such as colchicine and oryzalin (Assani et al., 2011), and by pollination of irradiated pollen in et al., 2003; Bouvier et al., 2002; Chalak and Legave, ‘Clemenules’ Clementine (Aleza et al., 2009). More- over, the morphological characteristics and the repro- ductive potential of the doubled haploid in Citrus and Received; May 22, 2014. Accepted; September 6, 2014. Published Online in J-STAGE. related species have not yet been reported. * Corresponding author (E-mail: [email protected]). Toolapong et al. (1996) selected haploid progeny
© 2015 The Japanese Society for Horticultural Science (JSHS), All rights reserved. Hort. J. 84 (1): 30–36. 2015. 31
of Agriculture, Shizuoka University, before being used for the experiment. The morphological characteristics of fully expanded leaves (i.e., leaf blade size, leaf weight per unit, guard cell size, and stoma density) and flowers just before bloom (i.e., sizes of flower bud, petal, pistil, ovary, and pollen, and numbers of petals, stamens, and ovules per ovary) were measured using 20 samples. The character- istics of open-pollinated fruit investigated were size, numbers of locules and seeds, soluble solid content (SSC), and titratable acidity (TA). For each measure- ment, 20 samples were used in the doubled haploid, 5 samples in the haploid, and 10 samples in ‘Banpeiyu’ pummelo. Fig. 1. The doubled haploid induced by colchicine-treated axillary Pollen fertility was evaluated by stainability and in shoot buds of a haploid plant from ‘Banpeiyu’ pummelo. A: 10-year-old doubled haploid tree (Bar = 30 cm). B: Flowers of vitro germination. Pollen stainability was estimated by the doubled haploid (Bar = 3 cm). C: Fruit of the doubled hap- staining the samples with 1% acetocarmine after loid (Bar = 10 cm). squashing nearly mature anthers on a slide glass. In vitro germination of the pollen grains was performed on microscope slides covered with a 2-mm layer of 1% among small seed-derived seedlings obtained from the (w/v) agar medium containing 10% sucrose. Five sta- cross between ‘Banpeiyu’ pummelo and ‘Ruby Red’ mens, each from a different flower, were rubbed on the grapefruit (C. paradisi Macfad.), and Yahata et al. agar medium, and the slides were then incubated for (2005a) confirmed that this haploid was derived from 10 h in a moistened chamber at 25°C in the dark. Each the female gamete of ‘Banpeiyu’ pummelo. After graft- test evaluated 1000 grains with five repetitions. ing onto the trifoliate orange [Poncirus trifoliata (L.) Raf.], it showed vigorous growth and flowered for the Crossing for evaluation of the reproductive potential of first time seven years after germination. However, it the doubled haploid was difficult to use this haploid for genetic analysis and The doubled haploid, the haploid pummelo, and 6 for planned breeding because it had no fertile female diploid species and cultivars of Citrus [‘Banpeiyu’ gamete and only a few fertile pollen grains (Yahata pummelo, ‘Kiyomi’ tangor (C. unshiu Marcow. × et al., 2005a, c, 2011). Therefore, Yahata et al. (2005b) C. sinensis Osbeck), ‘Miyauchi-Iyokan’ (C. iyo hort. ex produced the doubled haploid plant of this haploid Tanaka), Koji (C. leiocarpa hort. ex Tanaka), Hyuga- pummelo using colchicine-treated axillary shoot buds. natsu (C. tamurana hort. ex Tanaka), and Hassaku This doubled haploid showed vigorous growth com- (C. hassaku hort. ex Tanaka)] were used for reciprocal pared with the original haploid pummelo (Fig. 1A) and crosses. fortunately produced many flowers and fruit for the first The flowers were pollinated immediately after emas- time 5 years after top-graft onto trifoliate orange culation and covered with paraffin paper bags. Seeds (Fig. 1B, C). were collected from each fruit of the crosses at matu- In the present study, we investigated the morphologi- rity. The seeds were extracted from each fruit and cal characteristics of the doubled haploid induced by classified into two groups, namely, developed (normal colchicine-treated axillary shoot buds of a haploid plant development) and undeveloped (empty) seeds. The from ‘Banpeiyu’ pummelo. Furthermore, we evaluated seeds were then placed on moistened filter paper and the reproductive potential of the doubled haploid as a maintained at 25°C. After germination, the seedlings male or a female parent by crossing with diploid citrus were transplanted into pots containing vermiculite and cultivars. transferred to a greenhouse. Materials and Methods Ploidy level analysis Morphological characteristics of the doubled haploid A total of 30–50 seedlings per cross combination The haploid pummelo obtained from the cross be- were measured by flow cytometry, except for the cross tween ‘Banpeiyu’ pummelo and ‘Ruby Red’ grapefruit between ‘Miyauchi-Iyokan’ and the haploid. Young (Toolapong et al., 1996), the doubled haploid induced leaves were collected from their seedlings, chopped by colchicine-treated axillary shoot buds of the haploid with a razor blade in 2 mL of buffer solution containing plant (Yahata et al., 2005b) and ‘Banpeiyu’ pummelo 1.0% (v/v) Triton X-100, 140 mM mercaptoethanol, were used in the present study. These plant materials 50 mM Na2SO3 and 50 mM Tris-HCl at pH 7.5, and in- were grafted onto trifoliate orange, and maintained for cubated for 5 min according to the preparation method approximately 10 years in the greenhouse of the Faculty of Yahata et al. (2005a). Crude samples were filtered Table 1. Comparison of morphological characteristics of leaf in ‘Banpeiyu’ pummelo, the haploid and the doubled haploid. 32 M. Yahata, T. Nukaya, M. Sudo, T. Ohta, K. Yasuda, H. Inagaki, H. Mukai, H. Harada, T. Takagi, H. Komatsu and H. Kunitake
Table 1. Comparison of morphological characteristics of leaf in ‘Banpeiyu’ pummelo, the haploid and the doubled haploid.
Ploidy Leaf blade (cm) Shape index Leaf weight Guard cell (mm) Stoma density Strains z -2 -2 level Length Width of leaf blade (mg·cm ) Length Width (no.·mm ) ‘Banpeiyu’ 2x 18.3 ay 10.1 a 181.2 b 37.2 a 22.3 a 20.3 a 399.7 b Haploid x 14.5 b 5.3 b 273.6 a 21.7 b 17.1 b 15.1 b 683.6 a Doubled haploid 2x 17.9 a 9.4 a 190.4 b 36.7 a 22.5 a 20.4 a 442.4 b z (Length of leaf blade/Width of leaf blade) × 100. y Mean separation by Tukey’s multiple range test, P < 0.01. through Miracloth (Merck KGaA, Darmstadt, Germa- haploid (84.1% stainability and 32.9% pollen germina- ny) and stained with 25 μg·L−1 propidium iodide (PI). tion rate) was remarkably higher than that of the hap- The relative fluorescence of total DNA was measured loid (2.3% and 0.4%) (Fig. 3), but it was significantly for each nucleus with a Flow Cytometry System lower than that of ‘Banpeiyu’ pummelo (97.7% and (EPICS XL; Beckman Coulter, Fullerton, CA, USA) 88.2%). The fruit weight of the doubled haploid was equipped with an argon laser (488 nm, 15 mW). approximately 900 g, which was approximately half After flow cytometry analysis, chromosome observa- that of ‘Banpeiyu’ pummelo (Table 4; Fig. 2C). The tion was performed in 3 seedlings obtained from each doubled haploid produced a small number of locules cross combination. Root tips (approximately 5 mm compared with the ‘Banpeiyu’ pummelo. Furthermore, long) were excised from their seedlings, immersed in the number of seeds per fruit obtained from ‘Banpeiyu’ 2 mM 8-hydroxyquinoline for 24 h at 10°C, and fixed in pummelo was approximately 100, whereas that of the a mixed solution of ethanol and acetic acid (3:1) for doubled haploid was significantly less, approximately 24 h at 10°C. Enzymatic maceration and air-drying 60. The average size of the normal seeds from the dou- were performed according to the method of Fukui bled haploid was smaller than that of the seeds from (1996) with some modifications. Root tips were washed ‘Banpeiyu’ pummelo. On the other hand, there were no in distilled water to remove the fixative and then macer- significant differences in both SSC and TA of fruit juice ated in an enzyme mixture containing 2% (w/v) Cellu- among the haploid, ‘Banpeiyu’ pummelo, and the dou- lase Onozuka RS (Yakult Pharmaceutical Ind. Co. Ltd., bled haploid. Tokyo, Japan), 1% (w/v) Macerozyme R-200 (Yakult Haploids of fruit crops generally show poor growth Pharmaceutical Ind. Co. Ltd.), 0.3% (w/v) Pectolyase and their leaves, flowers, and fruit tend to be smaller (Kyowa Chemical Products Co. Ltd., Osaka, Japan), than those of diploid plants (Aleza et al., 2009; Chalak and 200 mM EDTA at 37°C for 20 min. The macerated and Legave, 1996; Dweikat and Lyrene, 1990; Höfer samples were rinsed with distilled water and a fixative and Lespinasse, 1996; Pooler and Scorza, 1995; solution was added. The mixtures were transferred to Toyama, 1974; Zhang and Lespinasse, 1991). On the glass slides. After the slides had been air-dried, the other hand, information on the morphology of doubled chromosomes were stained with 2% Giemsa solution haploids has rarely been reported for fruit crops, al- (Merck KGaA) in 1/30 M phosphate buffer (pH 6.8) for though doubled haploids have been produced in several 30 min, rinsed with distilled water, air-dried and ob- species, for example, kiwifruit, apple, banana, sweet served under an optical microscope (BX51; Olympus, cherry, peach, and Japanese pear (Germanà, 2006). Re- Tokyo, Japan). cently, some doubled haploids of apple were produced by in vitro androgenesis and in situ parthenogenesis, Results and Discussion and their morphology and reproductive potential have The morphological characteristics of the doubled been reported (Höfer et al., 2008; Okada et al., 2009; haploid were compared with those of the haploid and Vanwynsberghe et al., 2005), which showed that most ‘Banpeiyu’ pummelo. The sizes of leaf and guard cell of the doubled haploid apple lines had smaller leaves, of the doubled haploid were larger than those of the flowers, and fruit than the original diploid cultivars, and haploid and almost equal to those of ‘Banpeiyu’ pum- some of these doubled haploid lines also showed aber- melo (Table 1; Fig. 2A). Flower organ of the doubled rant morphology of flowers (Höfer et al., 2008; Okada haploid showed normal morphology (Fig. 2B). The et al., 2009; Vanwynsberghe et al., 2005). In the present flower and pollen of the doubled haploid were also study, on the other hand, doubled haploid pummelo significantly larger than those of the haploid, and no dif- showed normal flower morphology, whereas the hap- ference in flower and pollen size was observed in com- loid showed abnormalities such as the adhesion of pis- parison to those of ‘Banpeiyu’ pummelo (Tables 2 and tils and stamens in the flowers (Yahata et al., 2005a). 3). On the other hand, the doubled haploid had a signifi- Furthermore, this doubled haploid had significantly cantly reduced number of ovules per ovary, that is, ap- large leaf, flower, and fruit compared with those of the proximately half, compared with that of ‘Banpeiyu’ original haploid plant, and showed similar morphology pummelo. In addition, the pollen fertility of the doubled to that of ‘Banpeiyu’ pummelo. Table 2. Comparison of morphological characteristics of flower in ‘Banpeiyu’ pummelo, the haploid and the doubled haploid. Hort. J. 84 (1): 30–36. 2015. 33
Table 2. Comparison of morphological characteristics of flower in ‘Banpeiyu’ pummelo, the haploid and the doubled haploid.
Flower bud (mm) Petal (mm) Ovary (mm) No. of Ploidy No. of Pistil No. of Strains ovules per level petals (mm) stamens Length Width Length Width Height Diameter ovary ‘Banpeiyu’ 2x 31.5 az 16.7 b 4.2 30.5 a 15.5 a 19.4 a 7.4 a 7.5 a 206.1 a 43.3 a Haploid x 19.7 c 12.6 c 4.1 17.1 c 10.7 b 12.1 b 5.1 b 4.6 b 65.4 c 30.5 b Doubled haploid 2x 28.8 b 18.6 a 4.2 27.5 b 16.2 a 17.1 a 7.5 a 6.9 a 113.7 b 42.5 a Table 3. Comparison of morphological characteristics of pollen grain in ‘Banpeiyu’ pummelo, the haploid and the doubled haploid. z Mean separation by Tukey’s multiple range test, P < 0.01.
Table 3. Comparison of morphological characteristics of pollen grain in ‘Banpeiyu’ pummelo, the haploid and the doubled haploid.
Ploidy Pollen grain (mm) Shape index of Fertility (%) Strains z level Length Width pollen grain Stainability In vitro germination ‘Banpeiyu’ 2x 41.4 ay 26.5 a 156.2 a 97.7 a 88.2 a Haploid x 25.8 b 22.2 b 116.2 b 2.3 c 0.4 c Doubled haploid 2x 40.9 a 26.7 a 153.2 a 84.1 b 32.9 b z (Length of pollen grain/Width of pollen grain) × 100. y Mean separation by Tukey’s multiple range test, P < 0.01.
Fig. 3. Stainability by 1% acetocarmine (A, Bar = 50 μm) and in vitro germination (B, Bar = 50 μm) in pollen of the doubled haploid.
Fig. 2. The morphological characteristics of leaves (A, Bar = 5 cm), flowers (B, Bar = 3 cm), and fruit (C, Bar = 10 cm) in et al., 2009; Vanwynsberghe et al., 2005). The doubled ‘Banpeiyu’ pummelo (left), the haploid (center), and the dou- haploid pummelo in the present study also showed sim- bled haploid (right). ilar phenomena, although not severer than those of dou- bled haploids of apple; namely, the doubled haploid Some doubled haploid lines of apple showed low or showed lower pollen fertility and a smaller number of no pollen fertility and reduction of seed number com- seeds than ‘Banpeiyu’ pummelo. It was considered that pared with that of original diploid cultivars (Okada the reduction of the number of seeds was due to a de- Table 4. Comparison of morphological characteristics of fruit in ‘Banpeiyu’ pummelo, the haploid and the doubled haploid. 34 M. Yahata, T. Nukaya, M. Sudo, T. Ohta, K. Yasuda, H. Inagaki, H. Mukai, H. Harada, T. Takagi, H. Komatsu and H. Kunitake
Table 4. Comparison of morphological characteristics of fruit in ‘Banpeiyu’ pummelo, the haploid and the doubled haploid.
Fruit (cm) Shape No. of seeds % of Developed Soluble Titratable Ploidy Fruit No. of Strains index developed seed solid con- acidity level weight (g) locules Height Diameter of fruitz Developed Undeveloped Total seedsy weight (g) tent (%) (%) ‘Banpeiyu’ 2x 1691.5 ax 16.5 a 17.4 a 105.5 15.6 a 98.2 a 6.1 b 104.3 a 94.2 0.37 aw 10.9 1.3 Haploid x 205.5 c 9.1 c 9.4 c 103.3 10.5 c 0 c 0 c 0 c — — 11.1 1.1 Doubled haploid 2x 923.5 b 13.6 b 14.1 b 103.7 12.9 b 47.2 b 10.0 a 57.2 b 82.5 0.14 b 11.2 1.2 z (Diameter/Height) × 100. y (Developed seed/Total seed) × 100. x Mean separation by Tukey’s multiple range test, P < 0.01. Table 5. Fruit set and seed contents in the crosses among the doubled haploid, the haploid, and diploid cultivars. w Mean separation by t-test, P < 0.01.
Table 5. Fruit set, seed contents, and ploidy level of seedlings in the crosses among the doubled haploid, the haploid, and diploid cultivars.
Cross combination No. of seeds No. of Ploidy level No. of % of No. of No. of % of developed flowers developed seedlings fruit set fruit set seeds per Seed parent Pollen parent pollinated Developed Undeveloped seedsz examined 2x Others fruit Haploid Hyuga-natsu 10 0 0 — — — — — — — Hassaku 10 0 0 — — — — — — — Doubled haploid Hyuga-natsu 5 2 40.0 69 15 34.5 82.1 30 30 0 Hassaku 5 3 60.0 167 35 55.7 82.7 50 50 0 ‘Kiyomi’ ‘Banpeiyu’ 25 24 96.0 346 12 14.4 96.6 50 50 0 Haploid 25 20 80.0 62 4 3.1 93.9 30 30 0 Doubled haploid 25 23 92.0 434 11 18.9 97.5 50 50 0 ‘Miyauchi-Iyokan’ ‘Banpeiyu’ 10 9 90.0 83 6 9.2 93.3 40 40 0 Haploid 10 3 30.0 3 1 1.0 75.0 3 3 0 Doubled haploid 10 7 70.0 63 2 8.6 96.9 30 30 0 Koji ‘Banpeiyu’ 20 19 95.0 339 33 17.8 91.1 50 50 0 Haploid 20 1 5.0 0 0 0 — — — — Doubled haploid 20 15 75.0 256 7 17.1 97.3 50 50 0 z [Developed seed/(Developed seed + Undeveloped seed)] × 100. crease in the number of ovules per ovary. On the other hand, the cause of the decrease in pollen fertility is not clear. To account for the depression of pollen fertility in this doubled haploid pummelo, detailed cytological ob- servation, especially at meiosis, remains necessary. Fur- thermore, because the variations of morphological characteristics and reproductive functions have been observed among autotetraploid induced by colchicine Fig. 4. The seedlings obtained from the reciprocal crosses between treatment (Nukaya et al., 2011; Rêgo et al., 2011; Wu the doubled haploid and some diploid cultivars (A, Bar = 3 cm) et al., 2012), and given the influence of homogeniza- and the metaphase chromosomes in a root tip cell in one of the tion, it seems that further investigation of their aberra- seedlings (B, 2n = 2x = 18, Bar = 10 μm). tions in the doubled haploid is required. Additionally, because only one doubled haploid line was investigated in the reciprocal crosses between the doubled haploid in the present study, it will be necessary to produce a lot and diploid cultivars. These developed seeds germinat- of doubled haploids, for investigation of their general ed almost normally (Fig. 4A), and all of the seedlings reproductive functions in the future. had large wing leaves, which is a typical feature of the In order to evaluate the reproductive potential of the doubled haploid. FCM analysis of all those seedlings doubled haploid, crosses with the haploid and some showed that their fluorescence intensity coincided with diploid cultivars were carried out in the present study that of the diploid control. The chromosome count of (Table 5). In the reciprocal crosses between the haploid root tips revealed that all of the seedlings examined had and diploid cultivars, the haploid showed no fruit set as 18 chromosomes (Fig. 4B). a seed parent, and low fruit set and a few developed In apple, it was difficult to use the doubled haploid seeds as a pollen parent. On the other hand, when the lines as breeding materials because most of them had doubled haploid was used as the seed and/or pollen low and/or no reproductive potential, and no or only a parent, a lot of fruit and developed seeds were obtained few progeny were obtained in their cross combinations Hort. J. 84 (1): 30–36. 2015. 35
(Okada et al., 2009; Vanwynsberghe et al., 2005). In the Cao, H., M. K. Biswas, Y. Lü, M. H. Amar, Z. Tong, Q. Xu, J. present study, on the other hand, when the doubled hap- Xu, W. Guo and X. Deng. 2011. Doubled haploid callus loid was used as both seed and pollen parents, a lot of lines of Valencia sweet orange recovered from anther cul- ture. Plant Cell Tiss. Organ Cult. 104: 415–423. developed seeds and diploid progeny were obtained. Chalak, L. and J. M. Legave. 1996. Oryzalin combined with ad- This result showed that the doubled haploid pummelo ventitious regeneration for an efficient chromosome dou- has no problem in term of the reproductive potential of bling of trihaploid kiwifruit. Plant Cell Rep. 16: 97–100. female and male gametes. Cuenca, J., P. Aleza, L. Navarro, P. Ollitrault and C. Proteccio. In conclusion, the doubled haploid induced using 2013. 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