The Journal of Heredity 2001:92(1)
© 2001 The American Genetic Association 92:56-64
Anther Culture-Derived Regenerants of Durum Wheat and Their Cytological Characterization
ramaci-AltuntepeFrom the U.S. Department of Agriculture–Agricultural Research Service, Northern Crop Science Laboratory, Fargo, ND 58105.
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Abstract |
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Anther culture is being increasingly used in cereal crop improvement both as a source of haploids and for inducing new genetic variation. We studied the androgenetic ability and regenerability of 10 cultivars of durum wheat (Triticum turgidum L., 2n = 4x = 28; AABB), using three different growth conditions and four media. From a total of 86,400 anthers cultured, 324 plants were obtained: 248 green and 76 albino. Genotype, growth condition, and media significantly affected anther response and callus production; interactions were also significant. Green plant regeneration was influenced significantly by genotype and growth condition, as well as by genotype and growth condition interactions. Albino plant regeneration was significantly affected only by growth condition. Regenerants showed gametoclonal/somaclonal variation. Differences in morphology, growth habit, adult plant height, spike size, and development of spikes at nodes were observed. Mitotic and meiotic chromosomes were studied by conventional staining and fluorescent genomic in situ hybridization techniques. Chromosome numbers of the regenerants ranged from 14 to 70. All 76 haploid plantlets (2n = 2x = 14; AB) were albino. Some of the 28-chromosome regenerants were also albino. Chromosome number in the green plantlets ranged from 28 to 70. Chromosome number also varied in regenerants originating from the same callus. Both intergenomic and intragenomic multivalents were observed. An interesting feature was the preferential multiplication of B-genome chromosomes, which formed multivalents (trivalents, quadrivalents, and hexavalents). We observed several chromosomal abnormalities, which seemed to increase with the level of polyploidy. Translocations, dicentric chromosomes, chromatid exchanges, and Robertsonian translocations involving the A- and B-genome chromosomes were observed. Chromosome breakages resulting in centric and acentric fragments, and telocentrics were observed. Chromosome multiplication and structural aberrations induced during culture may constitute the bases of gametoclonal and somaclonal variations.
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Introduction |
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Cell and tissue culture techniques such as anther culture have attracted considerable attention as supplementary tools to cereal crop improvement (Vasil and Vasil 1994). Anther culture involves the induction of embryoid formation from immature pollen and subsequent regeneration of embryoids into plantlets. Since Ouyang et al. (1973) reported the in vitro regeneration of plants from pollen, the research on wheat anther culture has progressed rapidly. Anther culture-derived haploids have been used to produce homozygous lines, which accelerate breeding programs (Kasha et al. 1990). The application of modern methods of direct gene transfer into crop plants also depends on the development of efficient systems for regeneration of full plants from cultured cells and tissues (Vasil and Vasil 1994). The development of efficient systems for regenerating haploid callus cultures may enable direct gene transfer into durum wheat. Chromosome doubling after the incorporation of a transgene(s) should facilitate its stable incorporation.
Although earlier workers obtained low frequencies (0.7%) of green plants (Ouyang et al. 1973), the frequency of microspore embryogenesis in common wheat has been improved considerably (see Chu et al. 1990). Several factors are considered important for increasing the induction frequency of green plantlets from anther culture (see Hu 1997), including the genotype of the donor plant and environmental conditions under which these plants are grown (Sibikeeva and Sibikeev 1996). However, not much is known about the anther-culture response (i.e., the ability to induce embryoids from microspores) of durum wheat. Studies on durum wheat reported the production of some albino plants (Zhu et al. 1979). In one case, two green plants were obtained (Hadwiger and Heberle-Bors 1986), while in another study three green and two albino plantlets were regenerated from anther culture of durum wheat (Ghaemi et al. 1993). For anther culture of durum wheat to be useful, a large number of anther culture-derived regenerants must be obtained. The objective of this study was to investigate the effect of the genotype of the donor plant, its growth conditions, and media on anther culture-derived haploid and polyploid regenerants of durum wheat, and to characterize the numerical and structural changes in chromosomes of the regenerated plants.
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Materials and Methods |
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Ten Turkish cultivars (Gediz-75, Sham-1, Cosmidor, Kunduru-1149, Ege-88, ;alCakmak- 79, Diyarbakir-81, Fenike, Dicle-74, Kiziltan- 93) of durum wheat (Triticum turgidum L., 2n = 4x = 28; AABB) were used as anther donors. Each experiment was set up with these 10 genotypes, 4 induction media [BAC-1 (Marsolais and Kasha 1985), BAD-1, BAD-3 (Trottier et al. 1993), and M-42 (Kao et al. 1991) modified with 2 g/L fructose, 4 g/L glucose, 50 g/L sucrose, and 130 g/L ficoll], and 3 growth conditions for donor plants (greenhouse, growth chamber, and field). The 120 combinations (10 x 4 x 3 factorial) were replicated 36 times.
Anther Culture
Donor plants were grown under greenhouse (36 automatically timed 400 W sodium vapor lights on a 16-h photoperiod as a supplement to natural lighting; temperature was maintained at 20–23°C), growth chamber (8 cool white and 7 warm white fluorescent lights, 160 W each; 10 incandescent lights, 40 W each; 16-h photoperiod with 60% relative humidity; temperature 21°C), and field conditions. Immature spikes were collected when the awns first emerged from the flag leaf, placed in a beaker containing enough distilled water to cover the stem at or just below the node prior to the spike, covered with plastic wrap, labeled, and refrigerated at 4°C for 7 days. After cold treatment, spikes were sterilized with 70% ethyl alcohol (EtOH) for 1 min, followed by 1% sodium hypochlorite and 0.1% Tween 20 for 8 min, then rinsed with sterile double-distilled water four to five times.
Each spike was staged by squashing its centermost anther in 1% acetocarmine. Anthers containing microspores at the mid-uninucleate stage were cultured. Twenty anthers were cultured in each well of a six-well culture plate containing 5 ml of one of the four induction media. Cultures were sealed with parafilm and kept in an incubator at 28°C in the dark. Cultures were observed weekly for callus and/or embryoid development. Calli and embryoids (1–2 mm) were transferred to 60 mm x 20 mm petri dishes containing 10 ml of BAD-1-based medium that we modified (30 g/L sucrose, 17.5 g/L glucose, gelled with 8 g/L agar instead of the ficoll). Transferred cultures were kept under the earlier induction conditions for 7–10 days, then moved to an incubation room at 25°C, under two warm white and two cool white, automatically timed fluorescent lights (34 W each) with a 16-h photoperiod. After plantlet regeneration, the cultures were transferred to 100 mm x 20 mm petri dishes containing 25 ml of modified MS medium (2 g/L kinetin, 1 g/L IAA, and 30 g/L maltose instead of sucrose) (Murashige and Skoog 1962) and kept in the same incubation room. Plantlets at the three-leaf stage were transferred to peat pellets and kept under the same conditions to acclimatize. After 1 week they were moved to the greenhouse previously mentioned, and transplanted into 13 cm pots filled with Sunshine Mix No. 1 (Sun Gro Horticulture, Bellevue, WA), and grown to maturity.
Chromosomal Studies
Both somatic and meiotic chromosomes were studied using conventional and fluorescent genomic in situ hybridization (GISH) techniques according to procedures standardized earlier (Jauhar et al. 1999). Slides with well-spread metaphase chromosomes were kept at -80°C for up to one month, if necessary, for GISH analysis.
Fluorescent GISH was carried out by hybridizing the A genome with Triticum urartu Tum. genomic DNA (labeled with biotin-14-dATP, 100 ng/slide) and blocking the B genome with Aegilops speltoides Tausch genomic DNA (500 ng/slide) according to the method described in Jauhar et al. (1999). The chromosome preparations were counterstained with propidium iodide (PI) or 4',6-diamidino-2-phenylindole (DAPI) and the labeled DNA was detected using fluorescein isothiocyanate (FITC). Slides were visualized and images prepared according to Jauhar et al. (1999).
Data Analyses
Analysis of variance (ANOVA) was conducted using SAS computer software (SAS 1998). The data were analyzed as a 10 x 4 x 3 random effects factorial design with genotype, growth condition, and media as independent effects. Dependent effects were the number of anthers forming calli and/or embryoids (= anther response), total calli production, and plant regeneration from individual plates.
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Results |
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Anther Culture Response and Callus Production
Of the total of 86,400 anthers cultured from 10 genotypes, 424 anthers (0.49%) responded (Table 1). Microspores that responded to culture developed either into embryoids or formed calli, although in some cases both embryoids and calli were derived from a single anther (Figure 1A). Many of the anthers produced only a single callus, while others produced several calli. In some cases a complete lobe of an anther was filled with calli (Figure 1B). Free-floating calli were also observed (Figure 1C) resulting from development of microspores that had dehisced from the anthers. Callusing of the anther filament was common. While subculturing, filament calli (Figure 1C) were removed whenever found so they were not mixed with other calli. Such calli were easily identified because most were still connected with the filaments. When these calli became detached from anther filaments during growth in culture, they could be easily identified by their relatively low cell density (translucent) and loose cell adhesion.
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All three factors and their interactions had a highly significant effect on anther culture response. From the 424 anthers that responded, 559 calli were obtained, which gave 0.65 calli per 100 anthers (Table 1). Callus production from anthers was significantly affected by all factors and their interactions (Table 2).
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Plant Regeneration
Five to 6 weeks after culture initiation, calli and embryoids were transferred to differentiation medium (modified BAD-1) for shoot and root initiation (Figure 1D). Cultures were then kept in the dark at 26°C until differentiation took place, usually within 7–10 days. While some plantlets started developing chlorophyll during differentiation, others turned green only after additional exposure to light (Figure 1E). After 1–2 weeks, calli showed an abundance of rooted plantlets (Figure 1F), which were separated and subcultured. However, some plantlets never developed chlorophyll and remained albino (Figure 1G). Seven to 10 days later, the green plantlets (Figure 1H) were transferred to soil. In two cases, chimeric plants (green and albino tillers together) originated from the same callus, probably from different tiller (axillary) primordia (Figure 1I).
Once green plants were transferred to the greenhouse, variations in growth habits were evident. Induced polyploids (2n > 28 chromosomes) tended to be smaller than the parent tetraploid plant. Some plants matured more rapidly and developed spikes from the first node. In addition, several other morphological abnormalities were observed, such as multiple spikes per tiller. Some pentaploid plant regenerants had spike morphology similar to that of hexaploid wheat.
Of the 324 plants (0.38%) regenerated from 86,400 cultured anthers, 248 were green (0.29%) and 76 were albino (0.09%) (Table 1). Thus 76.5% of the regenerants were green and 23.5% albino. The genotypic effect on green plant regeneration was highly significant and the effect of growth conditions was significant. Genotype growth condition interaction was also highly significant for green plant production. However, for albino plant regeneration, only growth conditions were significant (Table 2).
Effect of Genotype
Significant genotypic differences on anther culture response were observed. The highest mean for anther response (1.28%) was obtained from Dicle-74, while Çakmak-79 gave the lowest mean (0.06%). Dicle-74 also yielded the highest mean number of calli and was significantly better than all other genotypes except Cosmidor.
Only 3 of the 10 genotypes produced green plants. Dicle-74 gave the highest mean number of green plants, and was significantly different from Cosmidor, but not from Diyarbakir-81. Albino plants were obtained from 7 of the 10 genotypes, but differences among them were not significant.
Effect of Growth Condition
Anthers from material grown under all three conditions responded to culture. Anthers from field- and greenhouse-grown materials responded to culture significantly better and produced significantly more calli than those from the growth chamber-grown material. Field-grown material also gave somewhat better anther response and callus production than greenhouse-grown material, but again the differences were not significant.
Green plants were obtained only from field-grown and greenhouse-grown material. Although the field-grown material yielded more green plant regenerants than the greenhouse-grown material, the differences were not significant. Albino plants were obtained from both greenhouse- and field-grown materials, but the differences between the two growth conditions were not significant.
Effect of Media
The best anther culture response was obtained on BAD-1 (mean = 0.0085). Anthers cultured on BAD-1 media produced the highest mean number of calli. M-42 gave the lowest mean of anther response, but it was not significantly different from BAD-3. Although M-42 gave the lowest callus production, it gave the highest mean number of green plant regenerants, followed by BAD-1 and BAD-3. Statistical analyses revealed no significant differences among the media in producing green plants (Table 2). Albino plants were obtained from anthers cultured on all four media. Overall, field-grown Cosmidor cultured on BAD-1 medium gave the best anther culture [chresponse (4.6%); field-grown Dicle-74 cultured on BAD-1 medium had the highest callus production (6.4%); and field-grown Dicle-74 cultured on M-42 medium generated the highest number of green plants (6.5%).
Chromosomal Studies
Thirty-one of the 76 albino plantlets were evaluated for chromosome number using conventional techniques. Seven plantlets were haploid (Figure 2A) and 24 were tetraploid (Figure 2C,D; Table 3). Several chromosomal abnormalities were observed in the tetraploid albino plants. These included telocentric chromosomes, dicentric chromosomes (Figure 2B), and other abnormalities such as lightly stained regions (Figure 2B).
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Of the 248 green plants, 102 were evaluated for chromosome number (Table 3). Of these, 52 plants were tetraploid (2n = 4x = 28), 22 pentaploid (2n = 5x = 35), 6 hexaploid (2n = 6x = 42), 1 nonaploid (2n = 9x = 63), and 1 decaploid (2n = 10x = 70). Aneuploid plants with 2n = 36–39 chromosomes (Figure 2E) were also obtained with various structural abnormalities, that is, telocentrics, acrocentrics, fragments, and sticky ends. Meiotic cells of anther culture-derived polyploids and aneuploids showed various pairing configurations such as trivalents, quadrivalents, pentavalents, and hexavalents, which were investigated using fluorescent GISH.
Fluorescent GISH proved to be an excellent technique for identifying the A- and B-genome chromosomes in both mitotic and meiotic cells. In mitotic metaphase cells, GISH elucidated intergenomic translocations, chromatid exchanges, and preferential multiplication of chromosomes of one genome. By using morphological landmarks, precise chromosome duplications were revealed. For example, the characteristic 4A·7B translocation (Naranjo 1990) was easily detectable. In meiotic metaphase I cells, GISH helped to identify inter- and intragenomic chromosome pairing configurations. However, reorganizations within the same genome could not be detected, which is a typical disadvantage of GISH.
Mitotic cells. Green plants with 28 chromosomes were normal in genomic constitution, with 14 A-genome and 14 B-genome chromosomes (Figure 2C,D). However, in some polyploid cells there was a preferential multiplication of B-genome chromosomes (Table 4). Figure 2E,F, for example, shows a pentaploid cell with 14 chromosomes of the A genome and 21 of the B genome.
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In addition to numerical abnormalities, various structural abnormalities were also detected. Robertsonian translocations (Figure 3E–G) were frequent (observed in
15% of the cells studied). Interstitial translocations were also observed, albeit rarely. Intergenomic nonsister chromatid exchanges, both terminal and interstitial, were observed (Figure 3H). Fragments of A-genome chromosomes were more frequent than those of the B genome.
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Meiotic cells. Some tetraploid green plants had a normal chromosome complement (2n = 28) and regular pairing at meiotic metaphase I with 7 A-genome bivalents and 7 B-genome bivalents (Figure 3A,B). Univalents and intergenomic ring quadrivalents (Figure 3M) also occurred in several tetraploid plants. Preferential multiplication of B-genome chromosomes was also detected in meiotic cells (Figure 3C,D). Intra- and intergenomic rod and ring bivalents, some heteromorphic, were observed. Many of the V-shaped trivalents were of the same genome. Some of these appeared to be heteromorphic (Figure 3J).
Clear terminal (Figure 3I) and Robertsonian translocations (Figure 3N) between the A- and B-genome chromosomes were recorded, but interstitial translocations were never observed in meiotic cells. Frying-pan trivalents involved both A- and B-genome chromosomes. Some of these frying pans were due to translocations (Figure 3K). Quadrivalents were almost always comprised of chromosomes of both genomes, and many were clearly due to translocations (Figure 3L). In addition to intergenomic ring quadrivalents (Figure 3M), higher multivalents, for example, pentavalents (Figure 3O) and hexavalents, were also observed.
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Discussion |
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Anther culture is being increasingly used in wheat breeding programs both as a source of haploids and new genetic variation. Defining the factors affecting anther culture of durum wheat could increase the use of in vitro techniques in durum wheat breeding. We studied the anther-culture ability of 10 cultivars of durum wheat, using different growth conditions and media formulations. Although all 10 genotypes showed anther-culture response and produced calli, significant genotypic differences were observed. The genotype of the donor plant is known to affect anther culture response in wheat (Moieni and Sarrafi 1995). Both nuclear and cytoplasmic control of anther-culture response has been observed in wheat, although the majority of the variation is reported to be caused by nuclear genes (Ekiz and Konzak 1991).
The low yield of haploid green plants from wheat anther culture is a serious problem (Zhou and Konzak 1992). Even after culturing 86,400 anthers in 10 durum cultivars we did not obtain a single green haploid plantlet, only albino haploids. Earlier studies have shown that the production of green plants from anther culture of cereal crops is under the control of complex genetic factors (involving both additive and nonadditive gene action) as well as some cytoplasmic factors (Ekiz and Konzak 1991). Albino plants of barley, for example, do not contain mature chloroplasts and there is a large-scale deletion of the plastid genome in the microspore-derived albino plants of rice (see Jahne and Lörz 1995). It may be assumed that both the nuclear and cytoplasmic genes are needed for the production of green haploid plantlets in durum wheat. The lack of or deficiency in the plastid genomes of microspores may well contribute to the development of albino haploid plantlets in durum wheat. Although the cultured microspores will presumably have intact nuclear genes, they may not always have the cytoplasmic factors to interact with and would therefore be albino. This hypothesis is supported by the fact that durum wheat x maize hybridizations produce only green haploids (Almouslem et al. 1998), presumably because they contain the full plasmon of the female durum parent. It may be possible to verify this hypothesis by culturing ovules to produce haploids. If cytoplasmic factors are essential for green haploid production, then gynogenetic haploids would be green.
Although we did not recover green haploids, we obtained green regenerants at different ploidy levels ranging from 2n = 28–70 chromosomes. Yet the green plant regeneration in durum wheat (0.29%) in our study was much lower than that obtained in bread wheat (3.2%; Kasha et al. 1990). In our experiment, 76.5% of the regenerants did develop chlorophyll. Thus the green;t3:;t3albino ratio was somewhat encouraging. However, it is difficult to explain the wide-spread occurrence of albino plantlets. Several studies have shown that donor-plant growth condition strongly influences the yield of pollen plantlets in bread wheat (Holme et al. 1999). The expression of genes for chlorophyll development is influenced by environmental factors, such as growth conditions of the anther-donor plants (Bjørnstad et al. 1989) and cold pretreatment of anthers (Konzak and Zhou 1991). We found that field-grown donor plants yielded relatively more green plants, probably due to more protoplasts present in anthers since the plants were grown in the field. Similar results were obtained by Bjørnstad et al. (1989).
The media formulations and culture conditions may also contribute to the production of albino plantlets. The accumulation of lactic acid and other organic compounds or metabolic by-products during culture may damage the organelles in primordial cells and result in the production of albino plantlets (Kao 1981). It is also possible that the chemical composition of the medium coupled with the culture conditions cause mutation of the gene(s) that controls chlorophyll development. The recovery of chimeric plantlets (Figure 1I) shows that such a presumed gene mutation occurred during the formation of tiller (axillary) primordia, so the mutant and the normal tiller primordia responded differently to chlorophyll development.
The culture media formulation is reported to be a major factor for anther culture response and induction of the development of green plantlets from microspores (Fadel and Wenzel 1990; Hu 1997). We observed a strong media effect on anther response and callus production. Anther response was better on BAD-1 and BAC-1 than on BAD-3 and M-42. BAD-1 also gave the highest callus production and M-42 the lowest.
Polyploidy is the most frequently observed chromosomal abnormality, although aneuploidy has also been observed frequently in cell cultures (Evans 1989). The ploidy level of our green durum regenerants varied from 2n = 28 to 2n = 70. Chromosomal changes involving multiplication of entire genomes or some chromosomes have been reported in callus culture of wheat (e.g., Wang and Hu 1985). An interesting observation we made was the preferential multiplication of B-genome chromosomes. Fluorescent GISH of polyploid cells revealed disproportionately more B-genome chromosomes than A-genome chromosomes. Thus the cells shown in Figure 2E,F each contain 14 A- genome chromosomes and 21 B-genome chromosomes. The preferential multiplication of B-genome chromosomes was further evidenced by the presence of intragenomic multivalents within the B genome. The preponderance of B-genome chromosomes may be related to their predominantly heterochromatic nature. Heterochromatin is known to replicate later than euchromatin. That is why B chromosomes (accessory or supernumerary chromosomes) undergo nondisjunction, the mechanism by which they multiply (Jones and Rees 1982). Certain genetic factors may have caused the preferential multiplication of B-genome chromosomes.
There appeared to be a relationship between polyploidy and the development of chlorophyll. The frequency of green plants increased with the increase in level of ploidy. Haploid plantlets with 14 chromosomes were all albino. Plants with 28 chromosomes were either albino or green. However, it is interesting that plants with more than 28 chromosomes were all green. It appears as if the development of chlorophyll was ploidy dependent. It is likely that multiplication of both nuclear and cytoplasmic factors in the polyploid regenerants were responsible for chlorophyll production. A ploidy-dependent gene expression has been elegantly demonstrated by Galitski et al. (1999).
Some of the pentaploid (2n = 35) durum plants with additional doses of B genome looked more like bread wheat. It is likely that an additional dose of the B-genome chromosomes had an effect similar to that of D-genome chromosomes. It would appear that extra doses of B genome "compensated" for the absence of the D genome in durum wheat. This may perhaps be called compensation at the genomic level. If this phenomenon does indeed occur, it is analogous to nullisomic-tetrasomic compensation observed by Sears (1966) at the chromosomal level.
We also observed several different types of structural abnormalities of chromosomes including telocentrics, dicentrics, terminal or interstitial translocations, Robertsonian translocations, lightly stained regions, chromatid exchanges, and centric or acentric fragments. Several such aberrations have been observed by earlier workers (Lee and Phillips 1988). The potential for genetic damage from the widely used hormonal herbicide 2,4-D has been demonstrated. Pijnacker and Ferwerda (1994) showed the effect of 2,4-D and sucrose on sister chromatid exchanges (SCEs) in cell cultures of T. aestivum, T. durum, T. dicoccum, and T. monococcum. The tetraploids, T. dicoccum and T. durum, had relatively high mean numbers of SCEs. Although we used very low doses of 2,4-D (2–8 mg/L) in our media, the chemical may have contributed to chromatid exchanges observed in our studies. Robertsonians obviously arise by spontaneous breakage (misdivision) and reunion of chromosomes at the centromere. Events leading to chromosome breakage and, in some cases, subsequent exchanges or fusions of broken ends may therefore be of fundamental importance.
Anther cultures leading to callus production often generate genetic variation called gametoclonal variation, which describes phenotypically variant plants regenerated from gametophytic cells. Although the basis of gametoclonal (and somaclonal) variation is not understood, in vitro induced chromosomal aberrations may have contributed to such variation (Marburger and Jauhar 1989). Somaclonal variation may prove to be of significance from the breeding standpoint. In China, at least 20 cultivars of wheat have been produced using tissue culture techniques (Hu 1997). These cultivars, with superior agronomic traits (high yields and wide adaptation) are reported to be cultivated on more than 1 million hectares (Hu 1997). Lu et al. (2000) continue to use somatic tissue culture and anther culture to induce and stabilize variation to improve wheat breeding efficiency in China. Thus the development of an efficient anther-culture protocol is important and may have breeding implications.
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Acknowledgments |
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M. Do
ramaci-Altuntepe was a research scholar in Dr. Jauhar's laboratory at the USDA-ARS Northern Crop Science Laboratory, Fargo, ND; she thanks the Scientific and Technical Research Council of Turkey for the award of a scholarship. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the USDA or imply approval to the exclusion of other products that also may be suitable. |
Footnotes |
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Address correspondence to P. P. Jauhar at the address above or e-mail: prem_jauhar{at}ndsu.nodak.edu.
Corresponding Editor: Reid G. Palmer
Received September 7, 2000
Accepted November 11, 2000
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