Progress 10/01/03 to 09/30/06
Outputs
This project focused on recovery of onion and melon doubled haploids (DH) to speed development of stable non-segregating lines for breeding of improved crops. The onion work used materials from Dr. M. Mutschler's breeding program. The general procedures were as follows: a) culture 3-5 mm long unpollinated flower buds on BDS medium; b) transfer embryos obtained to elongation medium; c) determine ploidy by flow cytometry; d) transfer diploids (10-20 pct) to soil; e) treat haploid plants with anti-mitotic agents (colchicine, oryzalin, APM); f) transfer treated plants to soil for later determination of ploidy; g) grow plants to bulbing, vernalize, allow to flower, and self-pollinate for recovery of DH lines; h) test DH lines in the field in Ithaca, NY. Following these procedures, we recovered gynogenic plants from several pungent yellow breeding lines and from 3 promising mild lines. Few plants were obtained from the red lines tested. For chromosome doubling, whole basal
explants of in vitro plantlets were more suitable than split explants. APM at 100 and 150 micromolar gave similar regeneration of treated explants (70-80 pct) and recovery of diploid plants (15-30 pct) as colchicine at 750 and 1000 micromolar. APM has the advantage of much lower mammalian toxicity. Diploids were recovered from gynogenic plants that remained haploid after colchicine treatment by inducing formation of somatic shoots on haploid flower buds; over 60 pct of the somatic shoots were diploid. Seeds were recovered from many diploid plants and grown in the field in 2005 and/or 2006. The DH lines were assessed for bulb weight, pollen fertility, fecundity, uniformity, etc. Some look promising for further use. The melon work used materials from Dr. M. Jahn's breeding program that segregated for resistance to gummy stem blight, powdery mildew and 2 viruses. The general approach was to do pollinations with gamma-irradiated pollen (250 Gray), harvest fruits 3-5 weeks later, surface
sterilize the seeds, and culture them in liquid E20 medium in the light. Viable embryos were recovered from a small percentage of seeds. Embryos from 56 seeds from 3 different genotypes were grown to plantlets on solid medium and then cloned by nodal or tip cuttings to increase the number of plants from each line. Flow cytometry showed that most of the plants were haploid or mixoploid. Several chromosome doubling treatments were tested, including in vitro treatment of tip or node explants with 500 or 1000 mg/l colchicine for times ranging from 3 hours to 7 days. Regrowth was better higher with tip explants and with exposure of less than 4 days. Some of the treatments have resulted in plants with fertile pollen and/or increase in ploidy. In vivo treatments with 500, 1000 or 5000 mg/l colchicine were less promising, with decreased survival, abnormal growth morphology, and little viable pollen produced. Hundreds of parthenogenic clones from the various treatments have been transferred to
the greenhouse for attempted self-pollination. About 10 fruits have been recovered to date. Initial tests of uniformity and disease resistance of the DH lines are currently in progress.
Impacts
Production of doubled haploid (DH) plants via tissue culture techniques may substantially speed development of stable lines with improved yield, quality or resistance to diseases. The benefit is especially great with biennial crops like onion or complex traits controlled by multiple genes. We have obtained large numbers of DH onion lines of different genotypes and provided them to the Cornell onion-breeding program. Hundreds of our DH plants have been grown in field trials in Ithaca in 2005 and 2006. The data from these trials will provide an excellent test of the actual value of DH from culture to crop breeding programs. Seeds from the best lines will be made available to seed companies for further testing and use in development of improved hybrids. Similarly, the DH plants from our melon work may permit recovery of stable lines with multiple disease resistances, thereby increasing yield and quality and reducing the need for chemical control measures.
Publications
- Lim, W., Falise, M., Molly Jahn, M., and Earle, E. 2007. Production of doubled haploid (DH) plants and DH lines of melon (Cucumis melo L.). 2006 Report of the Vegetable Breeding Programs, Cornell Vegetable Breeding Institute, pp. 20-26.
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Progress 01/01/05 to 12/31/05
Outputs
We worked on doubled haploids (DH) of two horticultural crops: onion and melon. The onion research included establishment of cultures from new genotypes and continuation of analysis of plants from cultures initiated in 2004 or earlier. YIXI is promising yellow pungent line developed by the Mutschler breeding program. Flower buds from 5 YIXI plants were cultured using our standard procedures. Buds from 2 plants were unresponsive, but 1.5-3.0 percent of the 1300 buds from the other plants produced gynogenic plantlets. These were checked for ploidy by flow cytometry. Spontaneous diploid plants were transferred to soil, and haploids were treated with colchicine to induce chromosome doubling. The breeding program is growing diploid/mixoploid plants in the greenhouse, vernalizing them to induce flowering, and conducting further evaluations. We also cultured flower buds from 12 YIXC gynogenic plantlets that were still haploid or mixoploid after colchicine treatment. Somatic
plantlets occasionally developed from basal callus of buds cultured on standard BDS medium, but more somatic plantlets developed on medium containing thidiazuron as the only growth regulator. Some somatic plantlets from mixoploid buds shifted to diploidy, supporting the use of reculture to convert mixoploids to diploids. In 2004 we set up flower bud cultures from 8 genotypes (3 mild, 3 red, 2 yellow pungent). The red lines had a poor gynogenic response. The other genotypes gave rise to almost 250 gynogenic plantlets, of which some 60 were transferred to the greenhouse. These materials are now in the hands of the breeding program. DH plants from the 2003 cultures (many from YIXC) were used in a large 2005 field trial testing their bulb size, pollen fertility, seed set, etc. In addition, a seed increase was done from some of the YIXC and YIXE DH lines that performed well in a 2004 Ithaca field trial. We also started molecular studies using SSR primers. This work seeks to determine
whether spontaneous diploids recovered from flower bud cultures are true DH or are of somatic origin. In the melon work, we recovered DH lines via parthenogenesis from several melon genotypes from the Jahn melon breeding program. These genotypes segregate for resistance to gummy stem blight, powdery mildew, as well as zucchini yellow and papaya ringspot virus. The general approach was to do pollinations with gamma irradiated pollen (250 Gray), remove fruits 3-5 weeks later, surface sterilize the seeds, and culture them in liquid E20 medium in the light. Most of the seeds do not contain embryos but viable embryos were removed from a small percentage of seeds. These embryos were grown to plantlets on solid medium and then cloned by nodal or tip cuttings to increase the number of plants from each line. Flow cytometry showed that almost all of these plants were haploid. Treatment of tips and nodal segments with colchicine (500 mg/l, 3h) converted all plants tested to date to diploidy or
mixoploidy. These materials are now being cloned further. Rooted plantlets will be transferred to soil and provided to the melon breeding program for evaluation in the greenhouse.
Impacts
Production of doubled haploid (DH) plants via tissue culture techniques may substantially speed development of stable lines with improved yield, quality or resistance to diseases. The benefit is especially great with biennial crops like onion or complex traits controlled by multiple genes. We have obtained large numbers of DH onion lines of different genotypes and made them available to the Cornell onion-breeding program. Hundreds of DH plants from our program were grown in a field trial in Ithaca this summer. The data from this trial, currently being compiled, will provide an excellent test of the actual value of DH from culture to crop breeding programs. Similarly, the DH plants from our melon work may permit recovery of stable lines with multiple disease resistances, thereby increasing yield and quality and reducing the need for chemical control measures.
Publications
- Lim, W., Falise, M., Jahn, M, and Earle E. 2006. Production of doubled haploid plants of melon (Cucumis melo L.). 2005 Report of the Vegetable Breeding Programs, Cornell Vegetable Breeding Institute, pp. 25-28
- Lim, W., Goldschmeid, P., Mutschler, M.A., and Earle E. 2006. Onion doubled haploids: laboratory work. 2005 Report of the Vegetable Breeding Programs, Cornell Vegetable Breeding Institute, pp. 50-58.
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Progress 01/01/04 to 12/31/04
Outputs
This year we focused on production and analysis of onion doubled haploids (DH) via gynogenesis. Large numbers of gynogenic plants were available from cultures established in 2003 from materials in the Cornell onion-breeding program. Most were long day Allium cepa (yellow or red; pungent or mild). Some were various generations of crosses of A. cepa and A. roylei, a related species with resistance to Botrytis leaf blight. Flow cytometry revealed that about 15 percent of the plantlets recovered from immature flower buds were diploid. Most of the others were haploid, requiring chromosome doubling treatments to produce DH plants. Treatment of basal explants from young haploid plantlets with colchicine (300 mg/l in liquid medium for 48 hours) gave high survival and doubling frequencies. Two other antimitotic agents (amiprophosmethyl and oryzalin) at concentrations of 100 micro-mol or more also increased the ploidy of the explants. These agents were not superior to
colchicine, but may be of interest because they have lower mammalian toxicity than colchicine. Some 800 plants were transferred to the breeding program, where they were grown to bulbing in the greenhouse. The majority of these were from the yellow pungent breeding line designated YIXC. The plants are currently being vernalized to induce flowering for seed production. Although seed recovery has often been a problem in DH work, we obtained viable selfed seeds from DH obtained in previous years, thus producing stable DH lines. Seeds from 17 of these were grown in the field in Ithaca, NY for increase and initial evaluations. In the summer of 2004, we cultured about 10,000 immature A. cepa flower buds in order to provide additional materials for the breeding program. The genotypes used included three mild lines with a desirable combination of high Brix and low pungency (a combination of traits difficult to stabilize by conventional breeding); three red lines for which good seed production,
a range of flowering times, and good disease resistance would be desirable; and two other advanced pungent yellow breeding selections (YIXM and NY15-14-49). Because the parental plants were grown outdoors in a very rainy summer, losses to fungal and bacterial contamination were substantial. Some 200 gynogenic plantlets have been recovered to date, primarily from the mild lines. Some of these have already been analyzed by flow cytometry. The spontaneous diploids have been transferred out of culture, and the haploids are being treated with colchicine to induce chromosome doubling. In addition, we have begun molecular comparisons of spontaneous diploids recovered in previous years to the parental plants, using RAPD and SSR primers. The goal is to determine whether diploids recovered from within flower buds are indeed DH or whether they could be derivatives of somatic cells. If they are of somatic origin, they are of little value for crop improvement. We also initiated work on recovery of
haploids from four melon genotypes with resistance to several pathogens, following the general procedures in our publication (Lotfi et al. 2003. Plant Cell Reports 21:1121.
Impacts
Production of doubled haploid (DH) plants via tissue culture techniques may substantially speed development of stable lines with improved yield, quality or resistance to diseases. The benefit is especially great with biennial crops like onion or complex traits controlled by multiple genes. We have developed improved culture techniques and have provided large numbers of DH onion lines to the Cornell onion-breeding program. Some of these have already been grown in the field for increase and initial evaluation. Others are being grown for seed for tests next year. This project provides a good test of the actual value of DH from culture to crop breeding programs.
Publications
- Alan, A.R., Brants, A., Cobb, E., Goldschmied, P.A., Mutschler, M.A., and Earle, E.D. 2004. Fecund gynogenic lines from onion (Allium cepa L.) breeding materials. Plant Science 167:1055-1066.
- Alan AR, Brants A, Cobb E, Mutschler MA, Earle ED. 2003. Utilization of doubled-haploid technique in developing onion inbreds. Allium Improvement Newsletter 13:34-36.
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