Morphological Characteristics of a Doubled Haploid Line from ‘Banpeiyu’ Pummelo [Citrus Maxima (Burm.) Merr.] and Its Reproductive Function

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 reproductive 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 characteristics 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.

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