Source: CORNELL UNIVERSITY submitted to
CROP IMPROVEMENT VIA TISSUE CULTURE AND GENE TRANSFER
Sponsoring Institution
State Agricultural Experiment Station
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0194376
Grant No.
(N/A)
Project No.
NYC-149353
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2002
Project End Date
Sep 30, 2009
Grant Year
(N/A)
Project Director
Earle, E.
Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853
Performing Department
PLANT BREEDING
Non Technical Summary
Diseases and insect pests cause serious crop losses in horticultural crops. Breeding for resistance can be difficult and slow. This project uses laboratory techniques such as tissue culture and gene transfer to produce plants with resistance to disease and pests more efficiently than is possible by conventional methods.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011420108015%
2011440108025%
2011451108020%
2151440108030%
2161440108010%
Goals / Objectives
Objectives are to use plant tissue culture and gene transfer technology to improve pest resistance in horticultural crops. Specifically this includes A) production of haploid and doubled haploid plants from onion, melon, and Brassicas in order to speed production of stable inbred lines with disease resistance and B) production of Bt transgenic Brassica oleracea lines for use as trap crops in cabbage production. Additional objectives are improvement of culture and gene transfer techniques in these and related crops.
Project Methods
The starting materials for work with onion are Allium cepa and A. cepa x A. roylei interspecific hybrids from the Mutschler onion breeding program. A. roylei is highly resistant to Botrytis leaf blight. Onion haploids will be produced by culture of immature flowers on suitable nutrient media. Doubled haploids will be obtained by brief colchicine treatment of explants from regenerated haploid plantlets. Ploidy of treated materials will be determined by flow cytometry. Diploid material is transferred to the Mutschler breeding program for further use. The melon materials are Cucumis melo hybrids carrying multiple virus resistances. Melon haploids will be produced by pollination of female flowers with irradiated pollen, followed by culture of seeds from 21-day-old fruits in liquid medium. Parthenogenetic embryos that develop in the seeds are excised and grown to plantlets. Colchicine treatments of rapidly growing shoot tips are followed by flow cytometry. Diploid and mixoploid materials are transferred to soil for further use in the melon breeding program. The Brassica materials are advanced generations of blackrot resistant broccoli lines initially obtained by protoplast fusion of B. oleracea with blackrot resistant B. carinata. The resistant somatic hybrids were repeatedly backcrossed with broccoli and/or selfed. It was noted that the progeny of resistant plants did not segregate for resistance at the expected ratios and thus did not demonstrate stable resistance. Isolated microspores from resistant plants are being cultured, using procedures that are effective in some other Brassica materials, in hopes of stabilizing the resistance for use in the Griffiths crucifer breeding program. Chromosomes of the resistant plants will also be examined to determine whether abnormalities in chromosome number or structure are related to the loss of resistance in many progeny. Insect-resistant materials will be produced by Agrobacterium tumefaciens-mediated introduction of cry1Ac and cry1C Bacillus thuringiensis genes into Brassica crops and other vegetables. In particular, we will produce Bt-transgenic collards lines that are resistant to diamondback moths (DBM) and other Lepidopteran pests for use as trap crops in cabbage fields. Cytoplasmic male sterile (CMS) Bt-collards that do not produce pollen will be obtained by sexual crosses of Bt-collards with CMS cabbage provided by collaborator Farnham. Insect tests, including comparisons of DBM oviposition and larval survival on conventional collards and cabbage and Bt-collard, will be done in collaboration with Shelton. We will also try to improve the efficiency of the gene transfer process through use of leaf explants in place of seedling explants.

Progress 12/01/08 to 12/01/09

Outputs
OUTPUTS: This project has focused on two general areas of work: 1) production of Brassica vegetables expressing on or more Bacillus thuringiensis (Bt) genes for resistance to Lepidopteran insects; 2) production of doubled haploid (DH) lines of onion and melon for more efficient breeding of improved lines. The Bt materials were used by the Shelton group for various entomological studies. Green Comet broccoli plants carrying a Cry1Ac Bt gene and a Cry1C Bt gene, both under control of a 35SCaMV promoter, were maintained by clonal propagation in vitro because they are triploid and hence not fertile. Some of the DH onion lines from this project produced impressively robust plants with large bulbs, which have been evaluated for their breeding potential by collaborator Martha Mutschler. With cooperation of the Bejo Seed Company, hybrids of promising DH lines were produced in 2008, using two different sterile lines as female parents. One female was a Spanish style long day onion; the other was a more typical Northeast pungent long day onion. In the summer of 2009, replicated trials of 18 hybrids produced in 2008 were conducted in Elba and Oswego, NY. Most hybrids produced with a DH male parent and a Spanish style female parent outyielded the relevant commercial controls and several had a higher percentage of large and jumbo bulbs. Hybrids using the NE pungent female parent were better than the controls in one location and had generally similar yields at the second location. Further grading for bulb storability and quality will be done in January 2010. Two graduate students of Mark Bridgen (a faculty member located at the Long Island Horticultural and Research Center) did their thesis research on ornamental plants in my laboratory. One completed an M.S. thesis on "In vitro culture and height control of ornamental Plectranthus". The other is working on interspecific hybridization and embryo rescue of Cleome sp.). Lab facilities were also used to teach a course (Plant Tissue Culture Laboratory) covering a broad range of tissue culture techniques (micropropagation, embryo and ovule culture, anther culture, protoplast isolation and culture, suspension cultures, transformation). I also provided advice to other faculty, postdocs and students about their tissue culture experiments with a variety of agriculturally important species, including rice (e.g. strategies for growing rice plantlets in vitro in order to evaluate root morphology). PARTICIPANTS: M. Farnham, P. Griffiths, and M. Jahn are no longer participants in this project. TARGET AUDIENCES: Seed companies interested both in the plant materials developed and in improved techniques for use in their own laboratories. Academic researchers interested in enhanced recovery of doubled haploids of onion and melon. Researchers working on the genomics of onion or melon. Government regulators and public interest groups concerned with the ecological safety of Bt-transgenic crops. Undergraduate and graduate students interested in learning plant tissue culture techniques and/or using them in their research projects. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
This project has generated both information and plant materials. Our publication about improved procedures for production of melon doubled haploids has been of interest to seed companies and academic institutions, as judged by the requests for detailed information and advice. The most promising doubled haploid onion lines are being advanced in the Cornell onion breeding program. Results to date indicate an increase in heterosis of DH hybrids over comparable commercial controls. This suggests that further work on production of DH lines will be of benefit to onion breeding. Seeds from 11 pungent DH lines are available on request to seed companies for testing as parents of hybrids. Nine Cornell students or visitors learned plant tissue culture techniques and others benefited from advice about how to apply such techniques to their research projects.

Publications

  • Lim, W. and Earle, E.D. 2009. Enhanced recovery of doubled haploid lines from parthenogenetic plants of melon (Cucumis melo L.) Plant Cell Organ and Tissue Culture 98:351-356.
  • Mutschler, M.A., Hyde, P., Southwick, S., Westerling, N. and Earle, E. 2009. Doubled haploid onion lines. 2008 Report of the Vegetable Breeding Programs, Vegetable Breeding Institute, p. 36-39.


Progress 12/01/07 to 11/30/08

Outputs
OUTPUTS: This project has focussed on two general areas of work: production of Brassica vegetables expressing one or more Bacillus thuringiensis genes for insect resistance and production of doubled haploid (DH) lines of onion and melon for more efficient breeding of improved lines. The Bt materials were used by the Shelton group for tests of effects of these plants on predators and parasitoids. Green Comet broccoli plants carrying a cry1Ac Bt gene and a cry1C Bt gene are being maintained by clonal propagation in vitro because they are triploid and hence not fertile. Data from the melon DH project was organized for publication, including recommendations for the most efficient strategies for recovering haploids and fertile DH plants. With cooperation from Bejo Seed Company, Martha Mutschler increased seed of some of the onion DH lines (mostly long-day pungent ones) and produced experimental hybrids using two sterile lines for the female parent. Twenty-one hybrids were tested in the field in the 2008 season with promising results. Some mild DH lines were also evaluated. Two graduate students of Mark Bridgen (Long Island Horticultural Research & Extension Center) are doing research on tissue culture propagation of several ornamental plants in my laboratory. I also consult with other faculty, postdocs, and students about their tissue culture experiments. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Seed companies interested both in plant materials developed and in improved techniques for use in their own laboratories. Academic researchers interested in recovery of doubled haploids of a variety of crops. Researchers working on genomics of onion or melon. Government regulators and public interest groups concerned with the ecological safety of Bt-transgenic crops. PROJECT MODIFICATIONS: M. Farnham, P. Griffiths, and M. Jahn are no longer active co-investigators on this project.

Impacts
This project has generated both information and plant materials. The improved methods for production of melon doubled haploids are of interest to seed companies seeking more efficient and reliable breeding strategies. The 29 doubled haploid onion lines with some level of seed increase are being used to evaluate the DH approach to onion improvement. Some have been made available to seed companies for further testing. They have also been used by researchers interested in the molecular biology of onion. Studies with the Bt-transgenic lines have provided important information in relation to environmental assessment/ecological safety of Bt crops. This includes the fact that Bt plants are less hazardous to parasitoids than conventional insecticides. Bt collards and Indian mustard from this project were used to demonstrate the potential of Bt trap crops in cases where the cash crop (e.g. cabbage) is not suitable or desirable for genetic engineering.

Publications

  • Cao J, Shelton AM, Earle ED. 2007. Sequential transformation to pyramid two Bt genes in vegetable Indian mustard (Brassica juncea L.) and its potential for control of diamondback moth larvae. Plant Cell Reports 27:479-487
  • Chen M, Zhao J-Z, Collins HL, Earle ED, Cao J, M. Shelton AM. 2008. A critical assessment of the effects of Bt transgenic plants on parasitoids. PLoS ONE 3(5): e2284 doi:10.1371/journal.pone.0002284
  • Chen M, Zhao J-Z, Shelton AM, Cao J, Earle ED. 2008. Impact of single and dual-gene Bt broccoli on the herbivore Pieris rapae and its pupal endoparasitoid Pteromaluus puparium. Transgenic Res. 17: 545-555
  • Lim W, Earle ED. 2008. Effect of in vitro and in vivo colchicine treatments on pollen production and fruit recovery on melon plants obtained after pollination with irradiated pollen. Plant Cell Tissue Organ Culture 95:111-124
  • Mutschler M, Hyde P, Southwick S, DeJong D, Earle L. 2007. Development of long day doubled haploid onion lines. 2007 Report of the Vegetable Breeding Programs, Cornell Vegetable Breeding Institute, p. 30.
  • Shelton AM, Hatch SL, Zhao J-Z, Chen M, Earle ED, Cao J. 2007. Suppression of diamondback moth using Bt transgenic plants as a trap crop. Crop Protection 27: 403-409


Progress 12/31/06 to 11/30/07

Outputs
OUTPUTS: This work has produced about 70 DH lines of melon derived from starting materials that segregated for resistance to four pathogens: gummy stem blight, powdery mildew, zucchini yellows virus, and papaya ringspot virus. Ample numbers of seeds (100-500) with good germination are available from almost of all of the lines. These materials are currently being screened for resistance to gummy stem blight. They may allow identification of lines with stable resistance to one or more of the pathogens. A Powerpoint presentation was given to participants at the Cornell Vegetable Breeding Institute field day on August 27, 2007, and handouts were distributed. Information about the work was also provided in the 2006 Report of the Cornell Vegetable Breeding Institute. PARTICIPANTS: Wansang Lim (postdoc) Matt Falise (technician) TARGET AUDIENCES: Seed companies interested both in plant materials from the program and interested in improved techniques for use in their own laboratories. Academic researchers interested in recovery of doubled haploids of a variety of crops. Researchers working on genomics of melon (and onion from earlier years of this project) who wish to make use of the simplified genetic systems provided by DH materials.

Impacts
This year's work focused on efficient recovery of doubled haploid (DH) lines from gynogenic melon plants and resulted in improved methods for obtaining such lines. The plants were previously obtained after culture of seeds from fruits that resulted from pollination with irradiated pollen. The starting materials, obtained from Dr. Molly Jahn's melon breeding program, segregated for resistance to four pathogens: gummy stem blight, powdery mildew, zucchini yellows virus, and papaya ringspot virus. The goal was to obtain lines with stable resistance to these pathogens. Sixty-three plants were recovered from 15 fruits of 3 different genotypes. Flow cytometry showed that 33 (52%) were haploid, and 30 (48%) were mixoploid. Cloning of the plants in vitro provided material for tests of chromosome doubling treatments. Tests of short-term colchicine treatments of 1.5 and 2 cm shoot tips or of nodes showed the best survival and chromosome doubling with 3 cm tips exposed to 500 m g/L colchicine for 3 hours. Chromosome doubling was assessed by flow cytometry and/or pollen counts. Seventy-two plants from the in vitro colchicine treatments were transferred to the greenhouse and self-pollinated (average of 4.6 pollinations per plant). Fruits were obtained from 18 (25%) of them. In most cases, seed number was well over 100 (up to 530) and germination over 90%. Nodal explants would be attractive material for chromosome doubling because many (5-10) nodes can be obtained from a single plant in vitro; however, the initial work indicated poor (<40%) regeneration from colchicine-treated node after 3 weeks. In contrast, regeneration of untreated controls was >80%. By applying a mixture of plant growth regulators (IAA, BA, ABA) plus silver nitrate to nodes after exposure to colchicine, regeneration rates comparable to untreated controls were achieved. The most promising colchicine treatment for use with the hormone supplements was 500 mg/L colchicine for 12-24 hours. Fruit recovery from plants obtained by treatment of nodes was about 25%. About 50 fruits were obtained, most from different original gynogenic plants. Fruit recovery from plants that had good pollen production on flowers formed in vitro approached 50%; thus such pollen counts could be used to identify the most promising plants to transfer to the greenhouse. The fruits obtained had ample numbers of seeds. When embryos were removed from the seeds, germination was 100% in most cases. Thus nodal explants can serve as convenient material for recovery of DH plants from haploid/mixoploid material. Germinated embryos from about 40 DH lines are currently being screened in the greenhouse for tolerance to gummy stem blights. Assays with the other pathogens will also be conducted. In summary, this project has helped identify improved conditions for recovery of DH lines from melon for use in development of materials that are more tolerant of major diseases.

Publications

  • Alan AR, Lim W, Mutschler MA, Earle ED. 2007. Complementary strategies for ploidy manipulations in gynogenic onion (Allium cepa L.). Plant Science 173:25-31
  • Lim W, Falise M, Jahn M, 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.
  • Mutschler M, Goldschmeid P, Mayton H, Hyde P, Alan A, Lim W, Earle L. 2007. Doubled haploid onion. 2006 Report of the Vegetable Breeding Programs, Cornell Vegetable Breeding Institute, pp 40-44.


Progress 01/01/06 to 12/31/06

Outputs
In 2006, this project focused on production of doubled haploid (DH) plants of melon. The starting materials, from Dr. M. Jahn's breeding program, segregated for resistance to four pathogens: gummy stem blight, powdery mildew, zucchini yellows virus, and papaya ringspot virus. Parthenogenic embryos were obtained by pollination with gamma-irradiated pollen. Fruits were harvested 3-5 weeks later. Seeds were removed, surface sterilized, and cultured in liquid medium in the light. Viable embryos were recovered from a small percentage of seeds. Embryos from 56 seeds of 3 different genotypes were grown to plantlets on solid medium and cloned in vitro to increase the number of plants from each line. Flow cytometry showed that most of the plants were haploid or mixoploid and thus not products of normal fertilization. Several chromosome doubling treatments were tested on the clones, 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 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, leading to decreased survival, abnormal growth patterns, and little or no production of functional pollen. About 200 parthenogenic clones from the various treatments have been transferred to the greenhouse for attempted self-pollination. To date, about 10 fruits have been recovered. Seeds extracted from these fruits were recently planted. Initial tests of uniformity and disease resistance of the DH lines are in progress. Many additional plants from the more recent colchicine treatments are either in soil or will soon be potted. An alternate approach to production of haploid DH cucurbit plants was also tested. Explants from immature unpollinated ovaries of melon, cucumber, and zucchini were cultured on media containing various combinations of growth regulators. Effective surface sterilization procedures were determined but, to date, but no plantlets have been recovered. In previous work, insect-resistant Brassica juncea 'Green Wave' (Indian mustard) plants carrying the Bacillus thuringiensis cry1Ac or cry1C genes were produced for possible use as 'dead-end' trap crops. Seeds from several cry1Ac T1 plants were assayed for segregation of kanamycin resistance, in order to identify homozygous transgenic lines that would be useful for production of hybrid plants carrying both Bt genes. The Earle lab assisted several other research programs with tissue culture and gene transfer experiments. Materials (tobacco cell cultures), equipment, and advice provided enabled a researcher from the Wilson group to confirm that a bacterial enzyme she was studying hydrolyzed callose. Collaborative work with the lab of Dr. Li Li on analysis of the cauliflower Or gene resulted in two publications.

Impacts
Doubled haploid (DH) techniques have the potential to speed breeding of stable lines with improved quality, yield, and disease resistance. We have produced large numbers of DH melon plants from breeding lines that segregate for resistance to various plant diseases. Use of these materials by melon breeders should accelerate development of lines with multiple disease resistances and reduced need for chemical control. The insect-resistant Bt Brassica plants we have produced are a valuable resource for insect control and assessment of resistance management strategies. Our Bt Indian mustard and collard lines are promising dead-end trap crops that link GM technology and biological control systems without the use of a GM cash crop.

Publications

  • Lu, S., Van Eck, J., Zhou, X., Lopez, A.B., O'Halloran, D.M., Cosman, K.M., Conlin, B.J., Paolillo, D.J., Garvin, D.F., Vrebalov, J., Kochian, L.V., Kuepper, H., Earle, E.D., Cao, J., Li, L. 2007. The cauliflower Or gene encodes a cysteine-rich zinc finger domain-containing protein that mediates high-levels of beta-carotene accumulation. Plant Cell (published online).
  • Li, L., Lu, S., Cosman, K.M., Earle, E.D., Garvin, D.F., O'Neill, J. 2006. Beta -carotene accumulation induced by the cauliflower Or gene is not due to an increased capacity of biosynthesis. Phytochemistry 67:1177-1184.


Progress 01/01/05 to 12/31/05

Outputs
This project includes several areas of work, some of which are also covered in other reports. We produced doubled haploid (DH) onion plantlets through culture of immature flower buds from various promising materials in the Mutschler's onion-breeding program. Large numbers of DH plants were returned to the breeding program for further evaluation, including an extensive 2005 field trial in Ithaca, NY. Populations of DH melon plants were developed through culture of the occasional parthenogenic embryos formed in seeds after pollination with gamma-irradiated pollen. The starting genotypes were hybrid materials from the Jahn melon-breeding program segregating for resistance to multiple diseases. See report 149410 for details of the DH work with onion and melon. Our concept of a Bt-transgenic trap crop was developed further through work with two plants that are more attractive than cabbage for diamondback moth (DBM) oviposition: glossy leaf collards (cv. McCormacks Green Glaze; MGG) and Indian mustard (B. juncea cv. Green Wave). We now have glossy and non-glossy leaf collards with good expression of a cry1Ac Bt gene or a cry1C Bt gene. Sexual crosses of these plants produced pyramided collard lines with both Bt genes. We also produced and characterized Indian mustard lines expressing the cry1Ac or cry1C genes. Pyramided Indian mustard plants with both genes were produced by transforming cry1C seedling explants with the cry1A construct. All of these plants give excellent control of standard DBM larvae. Lines expressing a cry1C gene also controlled cry1A-resistant larvae while cry1A lines controlled cry1C-resistant larvae. Seeds were recovered from the various collard and Indian mustard lines. A greenhouse cage test conducted by the Shelton group in Geneva, NY during the summer of 2005 showed that growing cabbage plants in association with Bt collards or Bt Indian mustard suppresses diamondback moth populations (3 DBM generations were monitored) and reduces leaf damage on the cabbage. See report 149455. We concluded that broccoli plants carrying a cry1Ab gene under control of the chemically inducible Pr-1a promoter from tobacco do not give sufficiently tight control of gene expression to be useful for resistance management purposes, although there was significant signal transduction to untreated leaves and heads. See report 149538. In previous work, we identified several tobacco lines carrying the Trichoplusia ni baculovirus enhancin gene or the Bt cry1Ac gene that showed only marginal to moderate effects on T. ni larvae. Hybrid progeny lines carrying both genes were produced by sexual crosses followed by selection of seedlings resistant to the two antibiotics associated with the Bt and baculovirus constructs. These materials have been provided to Dr. Ping Wang (Geneva, NY) for tests of their efficacy in controlling cabbage loopers resistant to Bt.

Impacts
Doubled haploid (DH) techniques have the potential to speed breeding of stable lines with improved quality, yield, and disease resistance. The hundreds of DH onion plants we have obtained via tissue culture were evaluated in a large-scale field trial this summer. We expect the data from this trial to provide the first real test of actual contribution of the DH approach to onion breeding. Similarly our DH melon plants should accelerate development of lines with multiple disease resistance. The insect-resistant Bt Brassica plants we have produced are a valuable resource for insect control and assessment of resistance management strategies. They have revealed problems in potential use of one type of chemically inducible systems for resistance management. Our Bt collard and Indian mustard lines are promising dead-end trap crops that link GM technology and biological control systems without the use of a GM cash crop.

Publications

  • Cao, J., Bates, S.L., Zhao, J-Z, Shelton, A.M., Earle, E.D. 2006. Bt protein production, signal transduction, and insect control in chemically inducible PR-1a/cry1Ab broccoli plants. Plant Cell Reports (published on-line http://dx.doi.org/10.1007/s00299-005-0091-4
  • Cao, J., Shelton, A.M., and Earle, E.D. 2005. Development of transgenic collards (Brassica oleracea L. var. acephala) expressing a cry1Ac or cry1C Bt gene for control of the diamondback moth. Crop Protection 24:804-813


Progress 01/01/04 to 12/31/04

Outputs
This project covers several areas of work, some of which are also covered in other reports. We produced hundreds of doubled haploid (DH) onion plantlets through culture of immature flower buds from materials in the Cornell onion-breeding program. Chromosome doubling procedures using colchicine, oryzalin and APM were compared. DH plants were returned to the breeding program for further evaluation, including field trials. See report 149410 for details. Our concept of a Bt-transgenic trap crop was developed further through work with two plants that are more attractive than cabbage for diamondback moth (DBM) oviposition: glossy leaf collards (cv. McCormacks Green Glaze; MGG) and Indian mustard (B. juncea cv. Green Wave). We produced MGG lines with good expression of the cry1Ac Bt gene and crossed them with previously developed MGG expressing the cry1C gene to produce pyramided lines. Indian mustard lines with cry1Ac, cry1C or both genes were also produced. All give excellent control of DBM. Seed production from the various lines has been completed or is in progress, and a field test is planned for the summer of 2005. See report 149455. We found that broccoli plants carrying a cry1Ab gene under control of the chemically inducible Pr-1a promoter do not give sufficiently tight control of gene expression to be useful for resistance management purposes. See report 149538. To see whether it was possible to protect cauliflower leaves against insects without producing Bt protein in the head, we created broccoli and cauliflower plants carrying the cry1C Bt gene under control of a putative light-inducible promoter (from the ST-L8I photosynthetic gene of potato). ELISA of leaf and head tissues of the primary transformants and progeny showed that the Bt protein was produced not only in green leaves and heads of broccoli, as expected, but also in heads of cauliflower, presumably free of chloroplasts. Progeny seedlings produced Cry1C protein in their roots, but not in their hypocotyls, both in the light and in the dark. These results indicate the promoter used does not prevent gene expression in non-green tissue. In previous studies, we identified several tobacco lines carrying the Trichoplusia ni baculovirus enhancin gene or the Bt cry1Ac gene that showed only marginal to moderate effects on T. ni larvae. To determine whether these genes have synergistic effects on insect control, we produced hybrid progeny lines carrying both genes. Initial screens for inhibitory effects on larval growth and development identified a promising hybrid tobacco line. In insect bioassays non-transgenic plants suffered severe defoliation with an average 15 percent mortality and a mean insect head capsule size of 1.2 mm. Plants carrying both genes caused complete or nearly complete mortality with little or no defoliation. Agrobacterium tumefaciens transformation of Brassica oleracea uses seedlings explants but leaf explants would be more convenient. In experiments using leaf explants from in vitro grown broccoli or cauliflower, we found that transgenic callus was easier to obtain than normal transgenic plants. Leaf-based Brassica transformation method needs further work.

Impacts
It has been proposed that onion doubled haploids (DH) obtained from tissue culture will speed breeding of lines with improved quality, yield, and disease resistance. Our procedures for obtaining DH have produced large numbers of plants that are now being evaluated in the Cornell onion-breeding program. These evaluations will be a good test of the actual value of the DH approach. The insect-resistant Bt Brassica plants we have produced are a valuable resource for assessment of resistance management strategies. Most recently they have revealed problems in potential use of chemically inducible systems for resistance management. Our Bt collard and Indian mustard lines are promising dead-end trap crops that link GM technology and biological control systems.

Publications

  • Alan, A.R., Brants, A., Cobb, E., Goldschmied, P.A., Mutschler, M.A. Earle, E.D. 2004. Fecund gynogenic lines from onion (Allium cepa L.) breeding materials. Plant Science 167:1055-1066
  • Alan, A.R., Brants, A., Cobb, E., Mutschler, M.A., Earle, E.D. 2003. Utilization of doubled-haploid technique in developing onion inbreds. Allium Improvement Newsletter 13:34-36.
  • Cao, J., Shelton, A.M., Earle, E.D. 2005. Development of transgenic collards (Brassica oleracea L. var. acephala) expressing a cry1Ac or cry1C Bt gene for control of the diamondback moth. Crop Protection (in press)


Progress 01/01/03 to 12/31/03

Outputs
We continued our program on producing of doubled haploid (DH) onion plants. In 2003, over 1,300 gynogenic plantlets were obtained from about 28,000 immature flower buds collected from Allium cepa, A. roylei, and A. roylei-derived plants from the Cornell onion-breeding program. The A. roylei-derived plants were advanced generations of progeny of crosses between A. cepa and A. roylei (resistant to Botrytis leaf blight). Flow cytometry showed that most of the plantlets recovered (85 percent) were haploid, while about 10 percent were spontaneous DHs. Culture of buds on medium containing antimitotic agents such as amiprophosmethyl, oryzalin, or colchicine at various concentrations caused only a slight increase in the percentage of spontaneous DHs. To induce chromosome doubling in haploids, we used the procedure that we have found to be most efficient: exposure of basal explants from haploid plantlets to colchicine (300-400 mg/L) in liquid medium for 48 hours. To date, 75 DH A. cepa plants and 1 DH A. roylei-derived plant have been transferred to soil and provided to the breeding program. We expect to recover additional DH A. cepa and A. roylei-derived plants from the doubling treatments. We have initiated work on microspore culture of Brassica oleracea. In previous work, we obtained lines resistant to blackrot (Xanthomonas campestris pv. campestris, Xcc) by protoplast fusion of susceptible B. oleracea with blackrot-resistant B. carinata (PI 199947), followed by multiple generations of sexual crosses. Progeny of resistant plants usually included fewer resistant plants than expected, possibly because of chromosomal problems. Microspore-derived plants should be homozygous for all traits and possibly more stable. Several dozen plantlets have been recovered from microspores of blackrot-resistant broccoli plants. These will be tested for resistance to Xcc. In the area of gene transfer, we have produced Indian mustard (Brassica juncea, Green Wave) plants expressing a cry1C gene from Bacillus thuringiensis (Bt). These plants are resistant to diamondback moths and will be tested as possible dead end trap crops for cabbage. We also made crosses between two types of tobacco plants, each of which contains a different transgene related to insect resistance. One type has an enhancin gene from Trichoplusia ni (Tn) baculovirus, which causes developmental delay of Tn; the other has weak expression of the cry1Ac gene from Bt. Progeny with both genes were obtained and assayed with Tn. A line that shows synergistic insect control was identified. We are using our efficient system for recovery of Brassica plants from leaf explants to produce large numbers of cry1ac + cry1C transgenic broccoli plants for resistance management studies. We are also testing the leaf regeneration procedure for transformation, as an alternative to use of seedling explants and especially for rapid introduction of a second transgene into a transformed line. Some transformants have already been recovered using this approach.

Impacts
Onion DH will speed breeding of lines with improved quality, yield, and disease resistance. Brassica microspore culture procedures may allow blackrot resistance to be stabilized and will be helpful in other aspects of crucifer breeding at Cornell. The enhancin plus Bt insect resistant systems may provide a novel way to help delay development of Bt resistant insect pests. Our efficient system for in vitro propagation of Bt transgenic broccoli has allowed us to produce hundreds of plants for use in resistance management trials conducted by our entomology collaborators in Geneva, NY. Successful leaf transformation will make it easier to introduce additional transgenes into a transformed line.

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 and E., Earle, E.D. 2003. Production of gynogenic plants from hybrids of Allium cepa L. and A. roylei Stearn. Plant Science 165:1201-1211.
  • Cao, J. and Earle, E.D. 2003. Transgene expression in broccoli (Brassica oleracea var. italica) clones propagated in vitro via leaf explants. Plant Cell Reports 21:789-796.
  • 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.
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