Progress 10/01/99 to 09/30/04
Outputs
Production of doubled-haploid (DH) lines may cut the breeding of onion inbreds from 10 to 4 years. The aim of the project is to improve the efficiency of recovery of DH lines using gynogenetic embryo induction in onion lines adapted to NYS. Over 20,000 unopened flowers were cultured on gynogenetic embryo induction media. Most gynogenetic plantlets were obtained within 3-4 months after culture initiation. The rates of gynogenetic across genotypes were variable (0.3 to 15 pct). We obtained over 700 gynogenetic plantlets from which ca. 400 developed into plants. Flow cytometry showed that 65-85 pct of the gynogenetic plants were haploid, 15-30 pct were diploid, and the remaining plants were tetraploid or mixoploid. To induce chromosome doubling to re-establish diploidy and fertility, 2-4 month-old haploid plants were treated with colchicine. Basal explants provided a higher regeneration rate than longitudinally cut explants. Rates of DH recovery increased as colchicine
concentrations increased from 100 to 400 mg/l, but plant regeneration from the treated explants was reduced. The rate of doubling substantially increased when treatments were performed in liquid medium for 48 hours. The majority of plants regenerated from explants treated with 200-400 mg/l colchicine for 48 in liquid medium had increased ploidy levels (25-50 pct diploid, 6-30 pct tetraploid, and 10-30 pct mixoploid for haploid and diploid cells). Over 100 diploid and mixoploid onions obtained via spontaneous or induced doubling have produced bulbs after being grown in the greenhouse during summer of 2002. These bulbs are being kept in cold storage and will flower in spring of 2003. The flowers will be examined to determine the rates of fecundity among the gynogenetic plants and selfed to produce seed and fix the doubled haploid lines for future use. To facilitate transfer of A. roylei derived resistance to boytris leaf blight (BLB) to onion, some of the F1 and BLB resistant BC1F1 and
BC1F2 selections from the sexual program were also used in the doubled haploid program. It is clearly more difficult to obtain gynogenetic plantlets from A. roylei-derived plants than it is from the A. cepa. The emergence of gynogenetic plantlets from A. roylei-derived plants required up to three times as long as that of A. cepa-derived plantlets. No gynogenetic plantlets were obtained from culturing flowers from F1 and F2 plants. However, most of the plants from advanced generations responded to gynogenetic induction and provided 1 to 15 plantlets. In contrast to the gynogenetic plants obtained from A. cepa lines, the majority of gynogenetic plants obtained from A. roylei-derived plants were diploids (77 percent). Nearly all of the gynogenetic plants obtained from BC1F2 plants produced vigorous plants, which each produced 2 to 5 bulb-like structures, rather than bulbs capable of drying down (a roylei trait). Most of the gynogenetic A. roylei-derived plants will flower in 2003. Four
gynogenetic plants from BC1F2 plants flowered summer of 2002, producing viable pollen. One of these plants produced about 25 seeds after self-pollination. Two publications are in preparation for submission Spring 2003.
Impacts
Efficient production of doubled haploid (DH) lines could speed onion breeding substantially by providing an array of inbred lines within 2 years. The lines would be fully inbred, and so provide greater uniformity for onion hybrids made using the DH lines. The lines may also be more vigorous than traditionally bred onion inbred lines. This project has demonstrated that DH can be obtained from a range of lines important to New York and U.S. onion production. DH lines generated may prove to be very valuable to onion seed companies and growers.
Publications
- No publications reported this period
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Progress 01/01/01 to 12/31/01
Outputs
Summer of 2001, we started additional haploid cultures, using over 35,000 unpollinated flowers from 5 different groups of onion lines. These included two types of A. cepa (YIX Stock E and mild lines) as well as genotypes selected for resistance to Botrytis leaf blight from crosses between A. cepa and A. roylei. The latter included BC1F1 BC1F2 and F2 individuals. To date, over 600 shoots have been obtained from A. cepa lines, the majority of which grew into plantlets in culture tubes. Ten and 26 shoots were obtained from BC1F1 and BC1F2 lines, respectively. To date, ploidy of 161 regenerated plants (149 from A. cepa lines, 3 from A. roylei derived BC1F1, and 9 from BC1F2) has been determined by flow cytometry of leaf tissues. Among the A. cepa lines, 109 plantlets were haploid, 37 were diploid, and 3 were mixoploid containing both haploid and diploid cells. One of the BC1F1 plantlets was haploid, 1 was diploid, and 1 was a mixoploid. All nine interspecific BC1F2
plantlets were diploid. Diploid plantlets probably result from spontaneous doubling of chromosomes early in embryo development. Diploid and mixoploid plants were transferred to soil and given to the onion breeding program for evaluation. Five different colchicine treatments were applied to 134 haploid plantlets to determine the most effective method of doubling chromosome number of haploids. To date, flow cytometry was done on 82 shoots derived from 27 regenerated explants. Nine of these explants produced at least one diploid shoot, with a total of 19 diploid shoots identified to date. Colchicine-induced diploids were obtained from 4 genotypes. Overall, the diploidization rate was about 11%.
Impacts
Efficient production of doubled haploid (DH) lines could speed onion breeding substantially by providing an array of inbred lines within 1-2 years. This project has demonstrated that DH can be obtained from a range of lines important to New York and U.S. onion production. DH lines generated may prove to be very valuable to onion seed companies and growers.
Publications
- No publications reported this period
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Progress 01/01/00 to 12/31/00
Outputs
In 1999, we initiated a project on production of onion doubled haploids (DH) from immature flower buds. We cultured >13,000 buds from 8 onion genotypes, including the F1 hybrid between Allium cepa and A. roylei. From these cultures, we obtained 547 embryos, 400 of which formed rooted plants. Regeneration was genotype-dependent, ranging from 0.4% to 16.4% of the buds cultured. Best regeneration was from A. cepa; regeneration from the F1 hybrid between A. cepa and A. roylei was poor. Medium or large buds responded better than small ones. The nuclear DNA content of 258 regenerated plants was determined by flow cytometry: 20 (8%) were diploid (either DH or escapes), 237 (92%) were haploid, and one was triploid. Assays for four isozymes did not provide useful information about the homozygosity of the diploid plants. Two chromosome doubling agents (colchicine and APM) were used at various concentrations on 222 haploid plants. The treatments reduced survival substantially.
Diploids were obtained from some of the surviving plants but this aspect of the procedure needs improvement. About 30 DH plants in soil were transferred to the onion breeding program for further use and evaluation. In 2000, >19,000 flower buds from 13 genotypes were cultured. These included red onion varieties, varieties with increased resistance to Botrytis leaf blight, and interspecific hybrids. To date, 583 embryos have been recovered, of which 351 grew into plants. Flow cytometry on 80 of the plants showed that 71 were haploid and 9 were diploid, similar to last year. Further flow cytometry and chromosome doubling is in progress.
Impacts
Efficient production of doubled haploid (DH) lines could speed onion breeding substantially by providing an array of inbred lines within 1-2 years. This project has demonstrated that DH can be obtained from lines important to New York and U.S. onion production.
Publications
- No publications reported this period
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Progress 10/01/99 to 12/31/99
Outputs
Efficient production of doubled haploid (DH) lines could speed onion breeding substantially by providing an array of inbred lines in less than a year. Previous work in other groups has indicated that onion haploids can more readily be obtained from the unfertilized egg cells within ovules than from anther culture. We have recently initiated a project on production of onion DH via culture of unpollinated flowers. To date, we have cultured over 13,000 flowers from 8 onion genotypes, including the F1 hybrid between Allium cepa and A. royleii, and have recovered over 500 shoots. Some shoots were obtained from every line but efficiency ranged from <1% to 7%. Rooted plantlets have been recovered from most of the lines. We are using flow cytometry to determine whether these plantlets are haploid or diploid. To date, ca. 10% of the 200 rooted plantlets tested are diploid. Isozyme assays (esterase) will show whether these diploids are homozygous (i.e., derived from the egg
cell rather than from somatic tissue). Most of the remaining plantlets tested by flow cytometry are haploid; <5 % are chimeral. We will apply chromosome doubling treatments to the haploids to obtain DH from them. DH plants obtained are being transferred to soil for evaluation and use in the Cornell onion breeding program.
Impacts
Efficient production of doubled haploid lines could cut time for development of onion inbred lines by 75 percent or more, accelerating the introgression of resistance and other desirable traits into materials adapted to NY state.
Publications
- No publications reported this period
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