The Journal of Heredity 2001:92(1)
© 2001 The American Genetic Association 92:81-83
Brief Communication |
Production of Wheat Doubled Haploids by Pollination With Job's Tears (Coix lachryma-jobi L.)
From the Graduate School of Integrated Science and Kihara Institute for Biological Research, Yokohama City University, 641-12 Maioka-cho, Totsuka-ku, Yokohama 244-0813, Japan.
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Abstract |
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Wheat (Triticum aestivum L.) haploids were produced by crossing with Job's tears (Coix lachryma-jobi L.) as the pollen parent. Pollination was followed by 2,4-D treatment, detached tiller culture, and embryo culture, as described for maize pollination. The frequency of embryo formation was similar to that obtained by crossing wheat with maize pollen. Job's tears is a perennial plant which forms several stalks and its pollen can be collected throughout the year when the plant is maintained in a controlled environment. Our results indicate that Job's tears can be used as the pollen parent for wheat crosses for haploid production without requiring synchronization of flowering dates.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Haploid production followed by chromosome doubling results in the creation of genetically pure lines within a short period of time. This is an important procedure to shorten the time for crop improvement. In addition, a set of double haploid lines is useful for analyses of quantitative trait loci (QTLs) (Cadalen et al. 1998; Yan et al. 1999).
In wheat, haploids can be produced by interspecific crosses. In this procedure, haploid embryos appear as a result of selective elimination of the alien chromosomes during embryogenesis. Hordeum bulbosum was first used as the pollen parent for this purpose (Barcley 1975). However, subsequent studies indicated that this species was inappropriate for wheat cultivars due to the presence of dominant Kr genes, which reduce crossability (Snape et al. 1979). When maize pollen is crossed with wheat stigma, the sperm nuclei fertilize the wheat egg (Laurie and Bennett 1986; Zenkteler and Nitzsche 1984). However, the maize chromosomes are eliminated at early embryonic stages (Laurie and Bennett 1989). When hexaploid wheat or durum wheat is crossed with maize pollen, the efficiency of haploid production is not affected by Kr genes (Almouslem et al. 1998; Inagaki 1997; Laurie and Bennett 1987, 1988b). Other species related to maize, such as Teosinte (Zea mays L. spp. mexicana) and eastern gamagrass (Tripsacum dactyloides (L.) L.), are alternative pollen donors for wheat haploid production (Li et al. 1996; Riera-Lizarazu and Mujieeb-Kazi 1993; Suenaga et al. 1998; Ushiyama et al. 1991).
Cytological studies have provided evidence for the successful fertilization and elimination of paternal chromosomes from hybrid zygote in sorghum (Sorghum bicolor (L.) Moench) and pearl millet (Pennisetum glaucum (L.) R. Br.) crosses, and thus these species are also possible pollinators for wheat haploid production (Ahmad and Comeau 1990; Inagaki and Mujeeb-Kazi 1995; Laurie 1989; Laurie and Bennett 1988a). Here we report a new potential pollinator, Job's tears (Coix lachryma-jobi L.). Job's tears is a vigorous perennial grass of the tribe Maydeae, subfamily Panicoideae of Poaceae. It includes both cultivated and wild plants of Southeast Asian origin. The plant has thick rosetting stems. The top of the stem divides into several branches, and the plant is about 60–100 cm high. The cultivated type (ssp. mayuen) is used as food and medicine in eastern and southern Asian countries. The wild species inhabits wet ground such as riverbanks and the flowers bloom in summer to autumn. One male inflorescence contains several florets, and thus collection of the pollen is easy.
In this study we describe the potential use of Job's tears as a novel pollinator for wheat double-haploid production.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Plant Material
Common wheat (Triticum aestivum L.) cv. Chinese Spring was used as the female parent. Wheat plants were grown in potting soil in a temperature-controlled greenhouse [10°C/23°C (min/max)]. Job's tears plants were collected from the Maioka River close to our institution and were transferred to pots. As a control, the maize line 919J, which was kindly provided by the Maize Stock Center, was used as the pollen parent. Both pollen parents were grown in a temperature-controlled greenhouse [18°C/27°C (min/max)] with a light:dark cycle of 16:8 h. Flowering of Job's tears was maintained by growing lateral buds.
Crossing
The size of the anthers of Job's tears is similar to that of maize anthers (5–7 mm) (Figure 1). Several anthers could be obtained from the flowering florets in the male inflorescence. Fresh anthers of Job's tears collected by forceps and placed on the palm of the hand readily dehisced and were used for fertilization of the emasculated wheat spikes. Maize pollen was used as a control to pollinate wheat, using the method described previously by Inagaki (1997).
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Detached Tiller Culture
After pollination, wheat tillers were cut off and cultured for 14 days in a solution containing sucrose (4 %) and 2,4-D (100 mg/l). They were incubated in a growth chamber set at a constant temperature of 22.5°C, with a 16:8 h light:dark cycle and 80–90% relative humidity. The liquid medium was regularly changed with fresh medium throughout the cultivation period.
Double-Haploid Production
Culture embryos, plant rescue, and chromosome doubling were performed according to the procedures described by Inagaki (1997). The seeds were removed from the spikes and immersed in 70% ethanol for a few seconds then sterilized for 10 min in a solution containing 1–2% sodium hypochloride and a few drops per liter of Tween-20. After rinsing twice with sterile water, the seeds were dissected on sterilized filter papers, and the embryos were examined under the stereoscopic microscope. The embryos were transferred onto Murashige and Skoog medium (M-5519 one pack per liter; Sigma Chemical Co., St. Louis, MO) with 2% sucrose and 0.6% agarose in plastic dishes. The embryo rescue procedure was performed under aseptic conditions on a clean bench. The embryos were cultured at 22.5°C in a room set with a light:dark cycle of 16:8 h. After about 1 month the new plants grown from the embryos were transplanted to potting soil and grown in a greenhouse set at 22°C with relative humidity of 80% and 16:8 h light:dark cycle.
The plant somatic chromosomes were examined in squashed preparations of root-tip cells stained with acetocarmine. To induce chromosome doubling, the plants were treated with colchicine solution [0.05% colchicine, 2% dimethyl sulfoxide (DMSO), 15 drops/l Tween-20] for 12–15 h in darkness at room temperature, rinsed with water, and transferred to soil. Self-pollinated seeds were obtained from the plants. The likelihood of embryo and haploid formation was estimated using the following formulas:
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These values were subjected to analysis of variance (ANOVA) after arc sine transformation.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Wheat florets crossed with the pollen of Job's tears bore caryopses at 14 days after pollination that were smaller than those obtained by self-pollination. The caryopses were filled with an aqueous solution instead of the solid endosperm normally found in the wheat, and some contained immature embryos (Figure 2). The obtained embryos grew well and 30% formed normal shoots and roots. We examined the somatic chromosomes of all six regenerated plants. The root-tip cells of these plants carried 21 chromosomes of wheat, indicating a complete elimination of the chromosomes of Job's tears (Figure 3). ANOVA results indicated that the frequencies of embryo and haploid formation were not significantly different from those obtained by crossing with maize pollen (Table 1). Plants were treated with colchicine and diploid plants were obtained. The seeds of the double plants showed 42 normal chromosomes of common wheat.
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Collection of pollen from Job's tears is not a straightforward process when compared to collection of maize pollen. However, Job's tears is a perennial plant that forms lateral buds, and thus pollen can be obtained throughout the year (Figure 1). In the present study, although only a few plants of Job's tears were grown in the greenhouse, we were able to cross the pollen whenever wheat flowered. Thus we were able to easily produce wheat haploids without synchronization of the pollen donor. Consequently Job's tears can be used as a suitable pollen donor for wheat haploid production using the wide-cross method.
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Acknowledgments |
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The authors thank Dr. M. Inagaki, Ministry of Agriculture, Forestry and Fishery Japan for technical advice about wide hybridization.
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Footnotes |
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Address correspondence to Hisashi Tsujimoto, at the address above.
Corresponding Editor: Prem P. Jauhar
Received January 13, 2000
Accepted August 30, 2000
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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