Progress 10/01/97 to 09/30/03
Outputs
This project has used gene transfer, protoplast fusion, and haploid technology to produce horticultural crops with improved resistance to pests or pathogens. Genes transferred via Agrobacterium tumefaciens include several Bt genes (cry1Ac, cry1Ab, cry1C) into broccoli, cauliflower, Chinese cabbage and rapeseed; enhancin (from Trichoplusia ni baculovirus) into tobacco and broccoli; endochitinase from Trichoderma harzianum into tobacco, tomato, broccoli and rapeseed; and the lytic peptide MSI-99 into tobacco and tomato. Levels of expression of the various genes and their efficacy against target pests or pathogens were assessed by molecular and biochemical methods and by bioassays. The Bt genes were highly toxic to several Lepidopteran insects including diamondback moths; enhancin delayed development of Trichoplusia ni in tobacco; endochitinase had some activity against the fungus Alternaria brassicicola in broccoli but not against Sclerotinia sclerotiorum; and MSI-99
reduced symptoms of bacterial speck (Pseudomonas syringae pv. tomato) in tomato. MSI-99 was active against various bacteria and fungi in vitro. We developed an efficient system for propagation of transgenic crucifers starting from leaf explants and showed that expression of the cry1C gene (but not the cry1A gene) often decreases after a year of in vitro propagation. Good progress was made in haploid technology for onion and melon. We obtained hundreds of plantlets from immature flower buds from various Allium cepa and A. cepa X A. roylei (source of resistance to Botrytis leaf blight) lines used in the Cornell onion-breeding program. Most of the A. cepa regenerants were haploids. Chromosome doubling to obtain doubled haploid (DH) lines was achieved by exposing basal explants to colchicine in liquid medium for 48 hours. Hundreds of DH plants have been returned to the breeding program for further use. Haploid melon plantlets were obtained by pollinating flowers with irradiated pollen,
culturing seeds from 3-week-old fruits in liquid medium, and then excising any developed embryos for further culture. DH plants were obtained by colchicine treatment of nodes from the haploid plants. Protoplast fusion was used to produce crucifer vegetable lines with enhanced disease resistance. B. rapa and B. oleracea selected for high resistance to softrot (caused by Erwinia carotovora) were fused to produce B. napus, which was then backcrossed to B. rapa to yield Chinese cabbage with improved tolerance to softrot. Fusion of B. oleracea with a B. carinata source of resistance to blackrot (caused by Xanthomonas campestris pv. campestris), followed by multiple generations of sexual backcrosses or selfs, produced blackrot-resistant broccoli. The resistant lines showed aberrant segregation for resistance, with higher transmission via the female parent than via pollen.
Impacts
Genetic resistance to diseases and insects can reduce pesticide use and related costs and hazards. The DH onion and melon plants obtained could speed development and release of lines with improved disease resistance and quality because they provide homozygous inbred lines in one step. The time savings is especially valuable in onion, a highly heterozygous biennial crop. Blackrot resistant lines initially produced by protoplast fusion may help reduce damage from this serious disease of crucifers. The gene transfer work has produced insect resistant materials, improved transformation methods, and information about the effectiveness of several putative pesticidal genes. Seeds of some of the plant materials used have been provided to other researchers or to seed companies for further testing or use.
Publications
- Alan, A., Blowers, A. and Earle, E.D. 2003. Expression of a Magainin-type antimicrobial peptide gene (MSI-99) in tomato enhances resistance to bacterial speck disease. Plant Cell Reports 22:388-396.
- Alan, A.R., Mutschler, M.A., Brants, A., Cobb, E. and Earle, E.D. 2003. Production of gynogenic plants from hybrids of Allium cepa L. and A. roylei Stearn. Plant Science 165:1201-1211.
- Lotfi, M., Alan, A.R., Henning, M.J., Jahn, M.M. and Earle, E.D. 2003. Production of haploid and doubled haploid plants of melon (Cucumis melo L) for use in breeding for multiple virus resistance. Plant Cell Reports 21:1121-1128. Tonguc, M., Earle, E. and Griffiths, P.D. 2003. Segregation distortion of Brassica carinata derived black rot resistance in Brassica oleracea. Euphytica 134:269-276.
- Zhao, J., Cao, J., Li, Y., Collins, H.L., Roush, R.T., Earle, E.D. and Shelton, A.M. 2003. Transgenic plants expressing two Bacillus thuringiensis toxins delay insect resistance evolution. Nature Biotechnology 21:1493-1497
- Cao, J., Brants, A. and Earle, E.D. 2003. Cauliflower expressing a cry1C transgene control larvae of diamondback moths resistant or susceptible to Cry1A, and cabbage loopers. J. New Seeds 5:193-207.
- Earle, E.D. 2002. Plant Genomics and Transgenics at Cornell University, with emphasis on Brassica plants expressing Bt genes. Second Joint Colloquium on Agricultural Biotechnology, Challenges and Prospects of Agricultural Genomics, Seoul University, Suwon, Korea, pp. 3-13
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Progress 01/01/02 to 12/31/02
Outputs
This project uses haploid technology, gene transfer, and protoplast fusion to help produce crops with improved resistance to pests or pathogens. Haploid and doubled haploid (DH) plants were obtained from materials from the Cornell melon and onion breeding programs in order to speed development of stable inbred lines. Flowers from melon (Cucumis melo L.) hybrids carrying multiple virus resistances were pollinated with irradiated pollen and cultured in liquid medium for 10 days. Parthenogenetic embryos were excised, cultured, and grown to 175 mature plants. This system was more efficient than excising parthenogenetic embryos directly from seeds. A few of the plants recovered were spontaneous DH and fertile, but flow cytometry showed that most were haploid. Shoots (167) from the haploids were treated with colchicine in vitro. Ten diploids and 100 mixoploids were recovered. The diploids and many of the mixoploids had a high percentage of stainable pollen. Some of them
have formed fruits. Progeny will be evaluated for virus resistance and uniformity. DH were also recovered from various pungent or mild onion (Allium cepa) breeding lines by culture of unpollinated flowers. Some (10-20%) of the plants recovered were spontaneous DH. Chromosome doubling was induced in others by treatment of basal explants of young plantlets with colchicine (100-400 mg/L, 48 hr). Flowers from crosses of A. cepa with A. roylei (a source of resistance to Botrytis leaf blight) also produced some gynogenic plantlets, most of which were spontaneous diploids. Advanced generations (BC1F1, BC1F2) were more responsive than earlier generations (F1, F1). The DH onion materials were transferred to the breeding program for production of bulbs, flowers, and selfed seed. We are culturing microspores of blackrot-resistant advanced materials derived previously from protoplast fusion of Brassica oleracea with B. carinata (resistant to blackrot, Xanthomonas campestris pv. campestris) in
hopes of stabilizing the resistance (a problem to date). We developed an efficient system for cloning broccoli and other B. oleracea vegetables in vitro starting from young leaf explants. A broccoli plant with high expression of two different Bt genes was propagated for a year; expression of the genes in 30 clones was then tested by ELISA. All clones maintained high expression of the cry1Ac gene but most showed significant decreases in expression of the cry1C gene. Seeds were obtained from broccoli and cauliflower transformed with a cry1C Bt construct under control of a light-inducible (green tissue-specific) promoter. Plants from these seeds will be used to compare Cry1C protein production in heads of cauliflower and broccoli with the Bt gene under control of constitutive (35S CaMV) or light-inducible promoters. A transgenic tobacco line that carried a baculovirus gene (enhancin) and showed enhanced resistance to cabbage looper (Trichoplusia ni) was crossed with six tobacco lines
with low expression of a cry1Ac Bt gene, and progeny were recovered. Progeny that carry both genes will be used in bioassays to determine whether the two types of insect control are synergistic.
Impacts
Genetic resistance to diseases and insects can reduce pesticide use and related costs and hazards. The DH melon and onion plants obtained could accelerate development and release of lines with improved disease resistance and quality because they provide homozygous inbred lines in one step. This time savings is particularly significant in onion, a highly heterozygous biennial crop. Brassica materials derived from previous protoplast fusion experiments (lines resistant to blackrot and softrot; Ogura and Anand CMS lines) are also being released directly to seed companies for further use in their programs. Enhancin may prove to be a useful transgene for insect control. The observed reduction of Cry1C protein expression in long-term clones indicates that transgene expression after vegetative propagation should be monitored carefully.
Publications
- Alan, A., and E.D. Earle. 2002. Sensitivity of bacterial and fungal plant pathogens to the lytic peptides, MSI-99, Magainin II, and Cecropin B. Molec Plant-Microbe Interactions 15:701-708.
- Alan, A., A. Brants, E. Cobb, M.A. Mutschler, and E.D. Earle. 2002. Production of doubled-haploid onions in Allium cepa breeding lines adapted to New York State. Proceedings 2002 National Allium Research Conference, Dec. 11-14, Pasco, WA, pp. 73-74.
- Cao, J. and E.D. Earle. 2003. Transgene expression in broccoli (Brassica oleracea var. italica) clones propagated in vitro via leaf explants. Plant Cell Reports (in press).
- Cao, J., A. Brants, and E.D. Earle. 2003. Cauliflower expressing a cry1C transgene controls larvae of diamondback moths resistant or susceptible to Cry1A, and cabbage loopers. J. New Seeds (in press).
- Cao, J., H. Ibrahim, J.J. Garcia, H. Mason, R.R. Granados, and E. D. Earle. 2002. Transgenic tobacco plants carrying a baculovirus enhancin gene slow development and increase mortality of Trichoplusia ni larvae. Plant Cell Reports 21:244-250.
- Cao, J., J.-Z. Zhao, J.D. Tang, A.M. Shelton and E.D. Earle. 2002. Broccoli plants with pyramided cry1C and cry1Ac Bt genes control diamondback moths resistant to Cry1A and Cry1C proteins. Theor. Appl. Genet. 105:258-264.
- Mutschler, M.A., E. Cobb, A.R. Alan, and E.D. Earle. 2002. Transfer of traits from A. roylei to A. cepa by sexual transfer and/or doubled haploid production. Proc. 2002 National Allium Research Conference, Dec. 11-14, Pasco, WA, pp. 75-77.
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Progress 01/01/01 to 12/31/01
Outputs
This project develops plant materials from protoplast fusion or gene transfer to help produce crops with improved resistance to pathogens and pests. Further work was done with advanced backcross/selfed progeny of somatic hybrids obtained by fusion of Brassica oleracea with B. carinata (resistant to black rot, Xanthomonas campestris pv. campestris). These progeny include plants that have high resistance to blackrot but show peculiar patterns of segregation for resistance. Some of the best plants were cloned in vitro to preserve their genotypes. Collaborative work on microspore culture of resistant plants did not produce uniformly resistant lines and suggested difficulties in transmission of resistance after microsporogenesis. Seeds of the resistant plants, as well as CMS Brassica lines previously developed in vitro, were released to breeders for further use. In work on insect resistance, detailed bioassays of two tobacco lines transformed with a baculovirus enhancin
gene confirmed that they increase mortality and delay development of cabbage looper (Trichoplusia ni) larvae, even though expression of the enhancin gene is low. The resistant lines will be crossed with tobacco identified as having low expression of a cry1Ac Bt gene, to determine whether the two types of insect control are synergistic. Several cauliflower lines, including Freemont, Candid Charm and Snow Crown, were successfully transformed with a 35S-cry1C Bt gene, using Agrobacterium tumefaciens and seedling explants. Broccoli and cauliflower were also transformed with a cry1C construct under control of a light-inducible (green tissue-specific) promoter. Cry1C protein production in heads of cauliflower with constitutive or light-inducible promoters will be compared.
Impacts
This project has produced some plant materials of direct value to plant breeders. Ability to transform cauliflower will permit tests of agriculturally important transgenes in this major vegetable crop. The enhancin work demonstrates that this novel transgene is worth studying further for its contribution to transgenic insect control. Plants that express Bt proteins only in leaves and not in non-green commercial products (e.g. cauliflower heads) may gain more public acceptance than plants with constitutive expression of Bt genes.
Publications
- Alan, A. 2001. Utilization of lytic peptide and avirulence genes for developing plants with broad spectrum disease resistance. Ph.D. dissertation, Cornell University. 232 pp.
- Brants, A., C.R. Brown, and E. D. Earle. 2000. Trichoderma harzianum endochitinase does not provide resistance to Meloidogyne hapla in transgenic tobacco. J Nematology 32:289-296.
- Brants, A. and E.D. Earle. 2001. Transgenic tobacco cell cultures expressing a Trichoderma harzianum endochitinase gene release the enzyme into the medium. Plant Cell Reports 20:73-78.
- Cho H.-S., J. Cao, J. Ren, and E.D. Earle. 2001. Control of Lepidopteran insect pests in transgenic Chinese cabbage (Brassica rapa ssp. pekinensis) transformed with a synthetic Bacillus thuringiensis cry1C gene. Plant Cell Reports 20:1-7.
- Jyoti, J.L., A.M. Shelton, and E.D. Earle. 2001. Identifying sources and mechanisms of resistance in crucifers for control of cabbage maggot (Diptera: Anthomyiidae). J Econ Entomology 94:942-949.
- Mora, A. and E.D. Earle. 2001. Resistance to Alternaria brassicicola in transgenic broccoli expressing a Trichoderma harzianum endochitinase gene. Molecular Breeding 8:1-9.
- Mora, A. and E.D. Earle. 2001. Combination of Trichoderma harzianum endochitinase and a membrane-affecting fungicide on control of Alternaria leaf spot in transgenic broccoli plants. Applied Microbiology and Biotechnology 55:306-10.
- Ren, J.-P., M.H. Dickson, and E.D. Earle. 2001. CC-14-1 and CC-18-2 progenies of Chinese cabbage derived from somatic hybridization for resistance to bacterial soft rot. HortScience 36:990-991
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Progress 01/01/00 to 12/31/00
Outputs
Protoplast fusion and gene transfer are being used to develop crops with improved resistance to pathogens and pests. Advanced backcross/selfed progeny (BC3/s4 or s5) of somatic hybrids obtained by fusion of B. oleracea with B. carinata (resistant to black rot, Xanthomonas campestris pv. campestris) were tested in the greenhouse and field. Plants that were resistant to needle wound inoculation as seedlings were also resistant as adult plants. Most lines segregated for high resistance but one appeared to be uniform for this trait. Seeds of the best materials were released to breeders for transfer to other crucifer vegetables (e.g., cabbage, cauliflower). Progeny of tobacco transformed with a baculovirus enhancin gene were assayed with cabbage loopers (Trichoplusia ni). Larvae on some transgenic lines showed delayed and/or abnormal development, indicating that the enhancin transgene may be promising for insect control. Several cauliflower lines are being tested for
transformability with Agrobacterium tumefaciens, using seedling explants and a cry1C gene from Bacillus thuringiensis. In vitro assays of plant pathogens with three lytic peptides (cecropin, magainin, MSI-99) showed that 0.005-0.030 g/ml effectively inhibited many types of bacteria. Higher concentrations inhibited the fungi Penicillium digitatum, Alternaria solani, and Phytophthora infestans. In tomato leaf disc assays, infection by A. solani and P. infestans was reduced or prevented when conidia and sporangia were pre-treated with lytic peptides (0.01 g/ml). Some transgenic tomato lines expressing MSI-99 had enhanced resistance to bacterial speck (P. syringae pv. tomato).
Impacts
This work has produced some plant materials of direct value to plant breeders. It has also demonstrated that a novel transgene (enhancin) may be useful in insect control and is worth studying further. Ability to transform cauliflower will permit tests of agriculturally interesting transgenes in this important vegetable crop. The work with lytic peptides provides information about the levels of these molecules needed in transgenic crops to effect control of pathogens.
Publications
- Mora, A. 2000 Agrobacterium-mediated transformation of Brassica oleracea var. italica and B. napus with a Trichoderma harzianum endochitinase gene to enhance resistance against fungal pathogens. Ph.D. Dissertation, Cornell University. 215 pp. Ren, J.P., M. H. Dickson, M.H. and Earle, E.D. 2000. Improved resistance to bacterial soft rot by protoplast fusion between Brassica rapa and B. oleracea. Theor. Appl. Genet. 100:810-819.
- Shelton, A.M., Tang, J.D., Roush, R.T., Metz, T.D. and Earle, E.D. 2000. Field tests on managing resistance to Bt-engineered plants. Nature Biotechnology 18:339-342.
- Tabashnik, B.E, Roush, R.T., Earle, E.D. and Shelton, A.M. 2000. Resistance to Bt toxins. Science 287:42.
- Zhao, J.-Z., Collins, H.L., Tang, J.D., Cao, J., Earle, E.D., Roush, R.T., Herrero, S., Eschriche, B., Ferre, J. and Shelton, A.M. 2000. The development and characterization of diamondback moth resistance to transgenic broccoli expressing high levels of Cry1C. Appl. Environ. Microbiol. 66:3784-3789.
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Progress 01/01/99 to 12/31/99
Outputs
Protoplast fusion and gene transfer are being used to develop crops with improved resistance to pathogens and pests. Work on development of crucifer vegetables with improved tolerance to blackrot (Xanthomonas campestris pv. campestris) continued. Resistant somatic hybrids were previously obtained by fusion of resistant B. carinata with rapid cycling Brassica oleracea (RCBo). These were repeatedly crossed with broccoli and selfed to produce progeny that were evaluated for resistance and good horticultural type in the field. The 1999 field trial identified lines that showed good uniformity for high resistance to blackrot. Limited quantities of seeds will be available soon. A similar strategy is being used to obtain crucifer vegetables resistant to Alternaria brassicicola. Fusion of RCBo with Sinapis alba produced resistant somatic hybrids; some progeny from backcrosses to Green Comet (GC) broccoli also showed resistance. Five BC3 plants had consistently higher
resistance (mean of 1.4-1.8 on a 0-5 scale) than GC (4.1) in detached leaf assays and also in seedling assays. Seeds were recovered from four of the five lines. These results indicate that the enhanced resistance to Alternaria is heritable and could be a source of improved control of this serious disease. In transgenic work, selfed progeny were recovered from broccoli plants transformed with an endochitinase gene from the biocontrol fungus Trichoderma harzianum. The progeny lines show statistically significant enhanced tolerance to Alternaria brassicicola. Control was comparable to that seen with sub-optimal levels of fungicide (Bayleton), but no synergism between the expression of endochitinase and fungicide was detected. In contrast, endochitinase-transgenic broccoli and rapeseed did not show enhanced tolerance to another fungal pathogen, Sclerotinia sclerotiorum. Selfed progeny were also recovered from tobacco plants containing one of several different baculovirus enhancin genes.
The survival and rate of development of Trichoplusia ni larvae on the enhancin-transgenic plants are being studied to assess this approach to insect control.
Impacts
This work is aimed at creation of vegetable crops with increased resistance to serious disease and insect pests. Use of such plants will reduce the need for chemical pesticides that are not only costly but also may threaten human health and the environment.
Publications
- Cao, J., Tang, J.D., Strizhov, N., Shelton, A.M. and E. D. Earle 1999. Transgenic broccoli with high levels of Bacillus thuringiensis Cry1C protein control diamondback moth larvae resistant to Cry1A or Cry1C. Molec. Breeding 5:131-141.
- Tang, J.D., Collins, H.L., Roush, R.T., Metz, T.D., Earle, E.D., and Shelton, A.M. 1999. Survival, weight gain, and oviposition of resistant and susceptible Plutella xylostella (L.) (Lepidoptera: Plutellidae) on broccoli expressing Cry1Ac toxin of Bacillus thuringiensis. J. Econ. Entomol. 92:47-55.
- Earle, E.D. and V. Knauf. 1999. Genetic Engineering in Brassica. pp. 287-313 In: Biology of Brassica Cenospecies (Gomez-Campo, C. ed.). Elsevier.
- Earle, E.D., T. Cardi, M.H. Dickson, L.H. Hansen, D.W. Heath, J.-P. Ren, and M., Sigareva. 1999. Contributions of protoplast fusion to improvement of Brassica crops. pp. 131-134 In: A. Altman, M. Ziv, S. Izhar (eds.) Plant Biotechnology and in Vitro Biology in the 21st Century. Kluwer Academic Publishers.
- Ren, J., Dickson, M.H, and Earle, E.D. 1999. Re-synthesis of B. napus by protoplast fusion to improve resistance to bacterial soft rot. Eucarpia Cruciferae Newsl.34:133-134.
- Mora, A. and Earle, E.D. 1999. Transformation of broccoli with a Trichoderma harzianum endochitinase gene. Eucarpia Cruciferae Newsl. 21:57-58.
- Sigareva, M., Jianping Ren, J., Earle, E.D. 1999. Introgression of resistance to Alternaria brassicicola from Sinapis alba to Brassica oleracea via somatic hybridization and back-crosses. Eucarpia Cruciferae Newsl. 21:135-136.
- Xue, Q., E. Kossi, H. Li, N. Zhang, J. Yang, S. McCouch. and E. D. Earle. 1999. Regeneration of plants from protoplasts of photoperiod sensitive genic male sterile rice (Oryza sativa L.). Euphytica 205:167-172.
- Keskitalo, M., Angers, P., Earle, E. and Pehu, E. 1999. Chemical and genetical characterization of calli derived from somatic hybridization between tansy (Tanacetum vulgare L.) and pyrethrum [Tanacetum cinerariifolium (Trevir.) Schultz-Bip.]. Theor. Appl. Genet. 98: 1335-1343.
- Cardi, T., Bastia, T., Monti, L, and E.D. Earle. 1999. Organelle DNA and male fertility variation in Solanum spp. and interspecific somatic hybrids. Theor. Appl. Genet. 99:819-828.
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Progress 01/01/98 to 12/31/98
Outputs
Protoplast fusion and gene transfer are being used to develop crops with improved resistance to pathogens and pests. Seeds of broccoli with improved tolerance to blackrot (Xanthomonas campestris pv. campestris) were released in 1998. A 1998 field trial identified advanced generations with even better resistance to blackrot; these lines will be made available soon. Resistant cabbage is also being developed. The resistant materials were obtained by fusion of resistant B. carinata with rapid cycling Brassica oleracea (RCBo), followed by crosses and selfs of the somatic hybrids and field selection for resistance. Fusion of RCBo with Sinapis alba produced somatic hybrids resistant to Alternaria brassicicola. Some progeny obtained after a backcross to broccoli also showed resistance. In the next backcross generation, 50% of the progeny had higher resistance than the backcross parent. The best BC2 plants will be used in further crosses and selections. Transgenic broccoli and
Chinese cabbage carrying a synthetic cry1C Bacillus thuringiensis (Bt) gene showed excellent control of standard diamondback moths (DBM) and DBM resistant to Cry1Ac protein. Transgenic plants with high expression of Cry1C protein also initially controlled DBM selected for moderate resistance to Cry1C protein; however, after continued selection on the transgenic plants, DBM populations that survived on these plants were recovered. Broccoli and tobacco containing a baculovirus enhancin gene were produced to test whether this gene aids in insect control. Progeny of transgenic tobacco and tomato with high expression of a Trichoderma harzianum endochitinase gene were evaluated for resistance to fungi and nematodes. They showed little or no improvement in control of the Northern root knot nematode (Meloidogyne hapla), Rhizoctonia solani, or Alternaria solani. Progeny homozygous for the transgene were recovered for possible use in production of plants carrying genes that may be synergistic
with endochitinase (e.g., glucanase). Transgenic tobacco suspension cultures released endochitinase into the medium. Broccoli expressing the endochitinase gene showed some enhanced control of Alternaria brassicicola. Transgenic tobacco and tomato plants homozygous for a bacterial avrD gene or lytic peptide gene are being tested for possible pathogen control.
Impacts
(N/A)
Publications
- Salon, P. and E.D. Earle. 1998. Chromosome doubling and mode of reproduction of induced tetraploids of eastern gamagrass (Tripsacum dactyloides L.). Plant Cell Reports 11:881-885.
- Mullen, J., G. Adam, A. Blowers, and E. Earle. 1998. Biolistic transfer of large DNA fragments to tobacco cells using YACs retrofitted for plant transformation. Molec. Breed. 4/5:449-458.
- Shelton, A. M., J. D. Tang, C. J. Perez, E. D. Earle and R. T. Roush. 1998. Importance of application technology on resistance management. Proc. 2nd Pacific Rim Conference on Biotechnology of Bacillus thuringiensis. pp. 224-244.
- Cardi, T. and E.D. Earle. 1998. A PCR-based procedure for early identification of male sterile Brassica oleracea cybrids with CMS "Anand" cytoplasm. Eurcarpia Crucifeae Newsletter 20: 53-54.
- Sigareva, M.A. and E.D. Earle. 1999. Regeneration of plants from protoplasts of Capsella bursa-pastoris and somatic hybridization with rapid cycling Brassica oleracea. Plant Cell Reports 18:412-417.
- Earle, E.D. 1998. Crucifer Vegetables with resistance to blackrot and Alternaria leaf spot. New York State IPM Publication 123:1-3.
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Progress 01/01/97 to 12/31/97
Outputs
Protoplast fusion and gene transfer are being used to develop improved crops. Seeds of broccoli with improved tolerance to blackrot (Xanthomonas campestris pv. campestris) will be released soon. They were obtained by fusion of resistant B. carinata with rapid cycling Brassica oleracea (RCBo), followed by backcrosses and selfs of the somatic hybrids and field selection for resistance. Fusion of RCBo with Sinapis alba produced somatic hybrids resistant to Alternaria. Progeny obtained by embryo rescue after a backcross to broccoli also showed resistance. The next backcross generation is now being tested. Somatic hybrids of RCBo + crucifers with virtual immunity to Alternaria (Camelina sativa, Capsella bursa-pastoris) were also obtained. Transgenic broccoli carrying a synthetic cry1C Bacillus thuringiensis gene showed excellent control of diamondback moths, including strains selected for resistance to Cry1C or Cry1A protein. Progeny of transgenic tobacco, tomato,
broccoli, and rapeseed with high expression of a Trichoderma harzianum endochitinase gene are being evaluated for resistance to fungi and nematodes. No enhanced control of the Northern root knot nematode (Meloidogyne hapla) was seen in greenhouse tests with the endochitinase transgenic tobacco. A bacterial avrD gene and lytic peptide genes were introduced into tobacco and tomato for possible pathogen control. Agrobacterium tumefaciens transformation of broccoli was more efficient with bacterial strain GV3101 than with LBA4404.
Impacts
(N/A)
Publications
- SIGAREVA, M. and E.D. EARLE. 1997. Transfer of an cold tolerant Ogura male sterile cytoplasm from broccoli to cabbage by protoplast fusion. Theor. Appl. Genet. 94:213-220.
- CARDI, T. and E.D. EARLE. 1997. Production of new CMS Brassica oleracea by transfer of 'Anand' cytoplasm from B. rapa through protoplast fusion. Theor. Appl. Genet. 94:204-212.
- HANSEN, L.N. and E.D. EARLE. 1997. Somatic hybrids between Brassica oleracea L. and Sinapis alba L. with resistance to Alternaria brassicae (BERK) SACC. Theor. Appl. Genet. 94:1078-1085.
- HEATH, D.W. and E.D. EARLE. 1996. Resynthesis of rapeseed (Brassica napus L.): a comparison of sexual versus somatic hybridization. Plant Breeding 115:395-401.
- HEATH, D.W. and E.D. EARLE. 1997. Synthesis of low linolenic acid rapeseed (Brassica napus L.) through protoplast fusion. Euphytica
- SIGAREVA, M. and E.D. EARLE. 1997. Intertribal somatic hybrids between Camelina sativa and rapid cycling Brassica oleracea. Eucarpia Cruciferae Newsletter 19:49-50
- SIGAREVA, M. and E.D. EARLE. 1997. Capsella bursa-pastoris: Regeneration of plants from protoplasts and somatic hybridization with rapid cycling Brassica oleracea. Eucarpia Cruciferae Newsletter
- EARLE, E.D. 1997. Crucifer Vegetables with resistance to blackrot and Alternaria leaf spot. New York State IPM Publication 121:1-3.
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Progress 01/01/96 to 12/30/96
Outputs
Efficient protoplast fusion and analysis procedures allowed transfer of a cold-tolerant Ogura male sterile cytoplasm (CMS) from broccoli to cabbage in one step, saving generations of backcrossing. Almost 250 fusion-derived plants from 5 cabbage lines were scored as diploid by flow cytometry and as CMS by PCR. All that have flowered to date are male sterile with normal cabbage morphology, and so are useful for hybrid production. Broccoli, cauliflower, and cabbage lines into which the #Anand# CMS was transferred by fusion were advanced and provided to seed companies under a biological materials release agreement. Development of blackrot-resistant broccoli from somatic hybrids containing a Brassica napus source of resistance is progressing. Some of the materials performed well in a small field trial in Cortland, NY. Two approaches were used for development of Alternaria-resistant Brassica vegetables: 1) production of new resistant B. oleracea + Sinapis alba somatic
hybrids in hope of achieving adequate female fertility; 2) somatic hybridization of B. oleracea with Camelina sativa or Capsella bursa-pastoris, both of which have virtual immunity to Alternaria. Progeny were obtained from transgenic tobacco and tomato plants with high expression of a Trichoderma harizanum endochitinase gene. These are being used in tests of resistance to fungal pathogens. Cell suspensions initiated from the transgenic tobacco release endochitinase into the medium.
Impacts
(N/A)
Publications
- EARLE, E.D., T.D. METZ, R.T. ROUSH, AND A.M. SHELTON. 1996. Advances in transformation technology for vegetable Brassica. Acta Horticulturae 407:161-168.
- FRARY, A. AND E.D. EARLE. 1996. An examination of factors affecting the efficiency of Agrobacterium-mediated transformation of tomato. Plant Cell Reports 16:235-240.
- HEATH, D.W. AND E.D. EARLE. 1996. Synthesis of Ogura male sterile rapeseed (Brassica napus L.) with cold tolerance by protoplast fusion and effects of atrazine resistance on seed yield. Plant Cell Reports 15:939-944.
- SIGAREVA, M. AND E.D. EARLE. 1996. Direct transfer of a cold-tolerant Ogura malesterile cytoplasm from broccoli to cabbage by protoplast fusion. Eucarpia Cruciferae Newsletter 18:42-43.
- HANSEN, L.N. AND E.D. EARLE. 1996. Studies of host specificity and potential usefor in vitro selection of Destruxin B from Alternaria brassicae. Eucarpia Cruciferae Newsletter 18: 112-113.
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Progress 01/01/95 to 12/30/95
Outputs
Protoplast fusion and gene transfer are being used to develop improved crops. Anefficient method for rapid fusion-mediated transfer of Ogura cytoplasmic male sterility (CMS) from broccoli into cabbage was developed, using g-irradiation to eliminate the broccoli nucleus. Almost 200 CMS cybrids from 3 elite cabbage lines are being evaluated in California. The mitochondrial gene controlling Ogura CMS (orf138) is also being transferred directly into the nucleus of fertile plants to see whether this can confer sterility. Broccoli (cauliflower) lines carrying the Anand CMS have been developed by three crosses of broccoli (cauliflower) to Anand CMS Brassica oleracea cybrids. Some of these lines showed good seed set and horticultural characters in field trials and are available to seedsmen as a resource for hybrid production, as is Anand CMS cabbage. Strong resistance to blackrot (Xanthomonas campestris pv campestris) is being transferred from B. napus to broccoli via repeated
backcrosses of a resistant napus-oleracea somatic hybrid. Some progeny from a fourth backcross are still resistant. An improved protocol for Agrobacterium-mediated transformation of tomato was developed. Four constructs carrying an endochitinase gene from the biocontrol fungus Trichoderma harizanum were transferred to tomato and tobacco. Some of the transgenic plants showed high levels of endochitinase. They and their progeny will be tested for enhanced resistance to a range of fungal pathogens.
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Progress 01/01/94 to 12/30/94
Outputs
Protoplast fusion and gene transfer are being used to alter the nuclear and organellar DNA of tomato and Brassica for development of improved lines. Thirty fertile cabbage lines were tested for regeneration from protoplasts; several highly regenerable ones were identified. A cold-tolerant Ogura male sterile cytoplasm was then transferred into one line via protoplast fusion. Many diploid CMS cabbage plants were selected by flow cytometry and PCR using CMS-specific primers within 6 months. Rapid cycling B. oleracea plants into which the Anand CMS had been introduced by fusion were evaluated for male and female fertility. Anand CMS progeny with good seed set are being backcrossed to broccoli and cauliflower to obtain seed for a field test. Somatic hybrids of rapid cycling B. oleracea + blackrot-resistant B. napus were backcrossed twice to broccoli or kale. Some of the progeny are highly strong resistant to blackrot. A construct carrying an endochitinase gene from the
biocontrol fungus Trichoderma harizanum was transferred to tobacco, tomato, and cabbage plants via Agrobacterium tumefaciens. The transgenic plants will be assayed to see whether they have enhanced resistance to fungal pathogens. Tomato gene transfer experiments demonstrated that the Fen gene, a member of the Pto gene family, is responsible for sensitivity to the insecticide Fenthion. Many of the parameters in Agrobacterium-mediated transformation of tomato were evaluated in order to optimize the transformation protocol.
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Progress 01/01/92 to 12/30/92
Outputs
Protoplast fusion technology was used to create novel and useful Brassica materials. Cold tolerant Ogura cytoplasmic male sterile (cms) B. oleracea cybrid lines were distributed to 17 seed companies for testing in hybrid production. Transfer of the improved cms to cabbage via protoplast fusion is in progress. New cold tolerant cms B. napus lines were created by fusion of B. rapa and B. oleracea. This improved cms was transferred to pak choi by sexual backcrosses. Resynthesis of B. napus from diploid progenitors selected for specific fatty acid composition yielded a promising line with high erucic acid and large seed size and another with only 3.6% total saturated fats and 3.5% linolenic acid. Fusion of rapid cycling B. oleracea and a B. napus line resistant to blackrot produced resistant somatic hybrids. Transgenic tomato lines were produced via Agrobacterium tumefaciens vectors carrying tomato DNA likely to carry genes for resistance to Pseudomonas syringae (from
map-based cloning in the Tanksley lab); these plants are being tested for resistance. Transgenic tomato cell lines were obtained by microprojectile bombardment with plasmid DNA or yeast artificial chromosome (YAC) DNA containing 50kb of tomato DNA. YAC bombardments may permit transfer of larger amounts of DNA into plant cells than current gene transfer techniques.
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Progress 01/01/88 to 12/30/88
Outputs
Evaluation of plants regenerated from petiole callus of hybrids between Solanum tuberosum and S. berthaultii (which carries trichome-mediated insect resistance) was continued. One of 5 clonal lines that had improved yield with desirable trichome traits in 1987 also showed superior performance in 1988. Selfed and backcrossed progeny of 55 regenerates were evaluated in the greenhouse for droplets on type B trichomes; several regenerated lines had more droplets than comparable control backcrosses. Progeny from 18 selected families were evaluated in the field for marketable yield, vine type, leafhopper resistance, and trichome traits. The results indicate that a period in culture may give material with desirable new genetic combinaitons, possibly through increased recombination. In vitro tuberization tests could distinguish lines with early and very late maturity but not mid-season or late ones. Plants and progeny were recovered from B73 and W182BN corn cultures for tests
of somaclonal variation. Genetic analysis of 3 novel corn phenotypes recovered from culture revealed 2 single gene recessives and complex inheritance of an adherent tassel type. A cauliflower line obtained from leaf protoplast culture showed earlier maturity and improved traits in replicated field trials. Atrazine resistant male-sterile broccoli was produced by protoplast fusion. Plants were regenerated with high efficiency from peduncle explants of 4 varieties (11 lines) of Brassica oleracea.
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Progress 01/01/87 to 12/30/87
Outputs
Plants were regenerated from petiole callus of hybrids between Solanum tuberosumand S. Berthaultii (which carries trichome-mediated insect resistance), and their clonal progeny were evaluated in field trials for yield and trichomes. Some lines had trichomes like the original hybrids but higher yields, suggesting that in vitro culture may have broken the usual linkage between insect resistance and poor yield. In vitro tuberization of parental and hybrid lines was assessed to determine whether it could predict field performance. Atrazine-resistant cauliflower lines developed by fusion techniques were resistant to high levels of atrazine in soil, but grew more slowly than sensitive controls. Calli were recovered from protoplast cultures of rapid cycling Brassica campestris. B. campestris lines resistant to amino-ethyl-cysteine were selected for use in fusion experiments. Analysis of mitochondrial DNA of B. napus lines synthesized by fusion revealed recombinant forms;
progeny of some male sterile lines with recombinant mtDNA showed further alterations. Plant regeneration from B. oleracea peduncles was achieved as a first step toward Agrobacterium-mediated transformation. Procedures for mutagenesis of B. campestris seeds with nitrosomethylurea were developed. Extensive attempts to select corn callus resistant to Helminthosporium carbonum race 1 toxin were unsuccessful but toxin effects on protoplasts from resistant and sensitive lines were characterized.
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Progress 01/01/86 to 12/30/86
Outputs
Work with tissue cultures and regenerated plants from various field and sweet corn inbreds and hybrids was continued. Some progeny of regenerated plants showed desirable agronomic characteristics, good combining ability, and promising performance in hybrid trials. Most plants from long-term cultures of cms-S and cms-T lines had reverted to fertility. Exposure of younger cms-T cultures to the insecticide lannate (methomyl) yielded many resistant male-fertile plants. Selection for resistance to the toxin from Helminthosporium carbonum race 1 and for aluminum tolerance was initiated. A friable embryogenic culture of W182BN X B73 is being used in attempts to recover tissue and plants from corn protoplasts. Protoplasts from Black Mexican Sweet corn and sorghum cell lines are being fused with corn leaf protoplasts carrying different cytoplasms to obtain hybrid recombinant cytoplasms for molecular studies. Plants are being regenerated from explants and cultures of hybrids
between commercial and wild potato to see whether culture-induced genetic recombination can aid in recovery of lines with both trichome-based insect resistance and good horticultural traits. Fusion and plant regeneration techniques developed for Brassica species have yielded many atrazine-resistant B. napus lines, some with recombinant mitochondrial DNA, as well as an atrazine-resistant cauliflower line.
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Progress 01/01/85 to 12/30/85
Outputs
Progeny of plants regenerated from scutellar cultures of corn inbred W182BN showed considerable somaclonal variation, including heritable changes of possible agronomic value. Culture-derived inbreds performed well in hybrid trials. Plants were regenerated from additional important inbreds (including B73, MO17, H99, and various sweet corn lines) for further tests of the relevance of somaclonal variation to corn improvement. W182BN regenerates showed many maternally-inherited shifts from CMS-S to fertility with loss of the S-1, S-2 mtDNA found in most CMS-S lines. The corn cultures are also being used in selection for resistance to pesticides and fungal toxins. Attempts to recover plants from cereal callus protoplasts are continuing, with emphasis on the role of ethylene and ethylene antagonists. Micro-injection of isolated mitochondria into CMS-T corn protoplasts is being tested. Work on the interaction of oat protoplasts with the phytotoxin victorin includes
fusion of sensitive and resistant protoplasts and use of ion-specific electrodes for measurements of K+ leakage after toxin treatment. procedures for in vitro manipulation of Brassica species have been developed, e.g. plant regeneration from protoplasts and explants of broccoli and cauliflower; production and purification of cytoplasts from B. campestris; protoplast fusion. An atrazine-resistant B. napus with novel mtDNA has already been obtained by fusion of B. campestris and B. oleracea.
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Progress 01/01/84 to 12/30/84
Outputs
Procedures for plant regeneration from scutellar cultures of corn have been developed and used to recover large numbers of plants. Progeny of some of these plants show heritable agronomically significant differences from the inbred parent. Selection for resistance to herbicides which affect callus growth is in progress. Cultures from corn lines with different levels of resistance to fungal pathogens have been established for use in studies of in vitro screening/selection for disease-resistance. We can now get excellent release of small meristematic protoplasts from our regenerable corn tissue cultures. The protoplasts survive for a week or more, change shape, show budding and regenerate walls. We are trying to induce division by culturing the protoplasts or initial callus under varied conditions. We have also obtained barley callus capable of long-term growth without hormones. It may be a useful source of dividing cereal protoplasts. Work on the interaction of
fungal phytotoxins with cereal protoplasts and callus was continued. Victorin, from Helminthosporium victoriae, was purified by HPLC and used both for structural studies (in Prof. Meinwald's lab) and for physiological studies with oat protoplasts. Victorin stimulates production of extracellular polysaccharides of mixed composition by sensitive protoplasts.
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Progress 01/01/83 to 12/30/83
Outputs
Several different in vitro studies with corn are in progress. Immature embryos from 7 experimental lines in the Cornell corn breeding program were cultured to see whether regenerable callus could be obtained. Embryos of RD 4509 formed somatic embryoids within a week and are still growing well after 5 months in culture. Some plantlets have been recovered. Immature embryos and established callus of cms-T and male-fertile W182BN corn were treated with MnC1(2) and ethidium bromide, mutagens that preferentially induce organellar mutations in lower eukaryotes. Ethidium bromide at 0.01-0.05 mg/ml was selected as most suitable treatment for mutagenesis. Mutagenized and control cms-T callus is being grown on medium containing Helminthosporium maydis race T toxin, to see whether mutagenesis increases the rate of change to mitochondrion-encoded toxin-resistance. Mutagenized excised embryos are being grown to maturity to see whether alterations in male-fertility and other
cytoplasmic mutations can be induced. An efficient procedure for isolating protoplasts from regenerable corn callus has been developed. These protoplasts are being cultured under a variety of conditions. They may be more likely to divide and form callus and plants than protoplasts from leaf mesophyll. Cultures from immature embryos of 3 oat and 5 barley varieties have been established for use in selection for resistance to fungal toxins.
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Progress 01/01/82 to 12/30/82
Outputs
Reliable procedures have been developed for regeneration of plants from long-term scutellar callus cultures of the important New York State corn inbred W182BN (in 4 different cytoplasms). Plants grown in the greenhouse have been pollinated and seeds obtained. The first plants form such seed have had the typical W182BN phenotype. Some progeny of corsses between culturederived and standards seed-derived W182BN appear to have increased vigor. Regenerated plants are being monitored for changes in pollen fertility and for changes in low molecular weight mitochondrial DNA. The small fast-flowering B(1) Brassica campestris line developed at Cornell appears to be excellent material for combined conventional and somatic cell genetics of a crop plant. Seeds of this line have successfully been grown to maturity and seed set on defined media In Vitro. The usual self-incompatibility is apparently not expressed in a high humidity culture environment. Attempts to obtain
regenerable callus and to culture protoplasts from a variety of fresh and cultured plant parts of B(1) and broccoli lines are also in progress. Filtrates from cultures of the alfalfa pathogen Verticillium albo-atrum have been prepared and are being tested against regenerable cultured alfalfa cells and aseptically grown seedlings. The aim is to determine whether a phytotoxin is present.
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Progress 05/01/81 to 12/30/81
Outputs
Scutellar cultures of corn inbred W182BN in N, T, C, and S male sterile cytoplasms were established and maintained on Murashige-Skoog medium with 5 mg/1 2,4-D. Plants were regenerated from the N, T, and S lines, grown to flowering, and evaluated for fertility. Work on improving rates of regeneration is in progress. These cultures are being exposed to fungal pathotoxins in order to select toxin- and disease-resistant cells and plants. They are also a good source of aseptic organelles for use in organelle transfer experiments. A friable rapidly growing cell line was selected from immature embryo cultures of Sorghum. Protoplasts of this cell line can divide. They are, therefore, being used in fusion experiments involving T cytoplasm corn mesophyll protoplasts. The fusion products should be able to divide and develop into intergeneric intercytoplasmic hybrid tissue. Procedures for cell and protoplast cultures of several Brassica species are being developed. The
cultured material will be used in attempts to obtain atrazine-resistant male-sterile material, either by selection for atrazine resistance in male-sterile lines or by fusion of protoplasts from male-sterile and from atrazine-resistant lines. Material with both of these cytoplasmically inherited traits would be valuable horticulturally and also useful for research on organellar genetics in higher plants.
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