Progress 10/01/99 to 09/30/04
Outputs
Using sorghum as the model plant, we found Agrobacterium tumefaciens-mediated transformation is three times more efficient than biolistic bombardment (proportion of transgenic calli to total immature embryos placed on the medium: 0.99 vs 2.87) in producing transgenic calli where the green fluorescent protein and the phospho-6-mannose isomerase (PMI) were used as markers in the latter and bar and chitinase were used as markers in the former. PMI appeared to be a very useful selectable marker and chlorophenol red assay was powerful to distinguish transgenic from non-transgenic plants by observing the color change (from red to yellow) in the medium. The current protocol using Agrobacterium-mediated transformation also was more economical and needed less time to produce transgenic plants than bombardment. Most of the transgenic plants has single copy or two-copy of the transgene, no silencing was observed as evidenced by western blot analysis. In trying to produce high
amylose wheat by reducing the starch branching enzyme II and endosperm-specific promoter, however, Agrobacterium-mediated transformation using green fluorescent protein (GFP) was not successful due to the toxicity of GFP on wheat callus cells. Wheat calli showing fluorescence failed to regenerate into plantlets. On the contrary, using creeping bentgrass as material, Agrobacterium-mediated transformation was successful where hva 1 and tlp genes were incorporated with GFP and PMI as markers for selection and cv Crenshgaw was the testing material. Both precise and imprecise integration of the T-DNA were observed. The right border sequences were intact more often than the left border sequences after T-DNA integration. In summary, Agrobacterium-mediated transformation was suitable for transformation of sorghum and creeping bentgrass but not for wheat. Both GFP and PMI are useful markers in selecting the transgenics.
Impacts
Transformation provides a quick and unique way to improve crop plants by introducing the target gene(s) into the recipient plants. Commercially available transgenic plants are known, such as Bt corn, Bt cotton, Bt potato, and Roundup Ready soybean. More recently, Monsanto has Roundup Ready bentgrass ready for release, also a transgenic spring wheat. Transformation is a powerful tool to supplement the traditional breeding programs by directly incorporating desirable genes. Once the legality matters are simplified and people realize that transgenic crops are no different from the traditional wide crosses, the transgenic crops will be accepted. However, caution should be taken as allergen may be involved in some cases.
Publications
- Genetically Modified Crops - their development, uses, and risks. 2004. Book published by Haworth Press. pp. 394.
- Transformaiton: a pwoer tool for crop improvement. 2004. Book chapter, in: Genetically Modified Crops. Haworth Press, pp.1-16.
- Sorghum transformation for resistance to fungal pathogens and drought. 2004. Book chapter, in: Genetically Modified Crops. Haworth Press, pp. 203-223.
- Genetic manipulation of creeping bentgrass. 2004. Ph. D. thesis (DL Fu). pp. 206.
- Genetically modified turfgrass: a critical review. 2004. Recent Res. Devel. Genet. Breeding, 1:317-347.
- Efficient genetic transformation of sorghum using a visual screening marker. 2004. Genome (in press).
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Progress 01/01/03 to 12/31/03
Outputs
Turfgrass provides multiple benefits to human and environment. Economically, turfgrass is one of the most important groups of crops in the U. S., representing a 40 billion business. Creeping bentgrass (Agrostis palustris Huds.) is extensively used for golf course putting greens. Agrobacterium-mediated transformation of bentgrass, ?Crenshaw? was accomplished using strain LBA 4404 in concert with a binary plasmid pUbiTLP, which carries a rice thaumatin-like protein gene (tlp D34) and a bar selection marker. The genomic integration of the tlpD34 gene was confirmed bu PCR amplification and then by Southern analysis. The expression of tlpD34 gene was further confirmed by western blotting. A total of 47 transgenic bentgrass lines were screened against dollar spot caused by Sclerotinia homoeocarpa. Some transgenic lines showed improved resistance to the pathogen in comparison to the control plants. At the same time, the hval gene, encoding the LEA 3 protein for drought
tolerance, also was introduced into creeping bentgrass using a ABRC3 derived inducible promoters (ABA2) and the gfp reporter gene. The heterologous expression of the HVA1 protein was shown in the T0 plants. The drought tolerance of T0 transgenic lines was assessed by visual scoring of leaf wilting and most transgenic lines showed significantly delayed leaf wilting compared to the control under greenhouse conditions. The inheritance of hva1 gene and its expression were further characterized in T1 generation. The heterologous expression of LEA3 protein genes could offer a potential solution in lessening the impact of drought on turfgrass growth.
Impacts
It is always desirable to use new and improved techniques in producing transgenic plants for commercial use. Our technique currently in use will save time, labor, material, with a higher frequency to produce transgenic sorghum plants carrying the target gene, the TLP gene, which provides resistance to stalk rot and tolerance to drought. The new technique is able to be patented.
Publications
- 1. D. L. FU. 2003. Genetic manipulation of creeping bentgrass. Ph.D. thesis, 254 pp.
- 2. P. Ouyang. 2003. Genetical transformation of wheat . MS thesis, 88 pp.
- 3. SKINNER, D. Z., S. Muthukrishnan, and G. H. Liang. 2003. Crop transformation, a powerful tool for modifying crop plants. Book chapter, In: Genetically Modified Crops ? their development, uses, and risks. Haworth Press, NY (In press).
- 4. G. H. Liang, M. R. TUINSTRA, S. MUTHUKRISHNAN. 2003. Sorghum transformation for resistance to fungal pathogens. Book chapter, In: Genetically Modified Crops ? their development, uses, and risks. Haworth Press, NY (in press).
- 5. GAO, Z. S., H. W. CAI, AND G. H. LIANG, 2003. Assay of a recessive gene for resistance to Southern leaf blight in maize. Jour. Plant Breeding (submitted)
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Progress 01/01/02 to 12/31/02
Outputs
To transform bentgrass, wheat and sorghum. Agrobacterium is a superior technique than biolistic bombardment. It produces transgenic plants at a higher frequency and low copy numbers of the target gene, thus reducing the chance of being silenced. To produce transgenic plants more efficiently, we made gene constructs with only the reporter gene (GFP) and the target gene (TLP, G11) and remove the selectable markers, bar and cah, which are deleterious to the calli growth and seedling regeneration. In addition, instead of obtaining transgenic plants in 8-9 months, we could produce transgenic plants within 3 months. The new technique using Agrobacterium as a transformation tool works for inbred lines as well as a commercial sorghum hybrid, suggesting that it is reproducible and applicable to various genotypes. Likewise, Agrobacterium transformation also works for wheat cultivars using the transgene, PLD for frost tolerance and Dx, Dy genes for better baking quality.
However, the prerequisite of the technique still is tissue culture, that is, the plant material must be amenable to tissue culture manipulations ? callus induction and plant regeneration. Transformation techniques that is able to by-pass the tissue culture step is very desirable and deserve additional research.
Impacts
It is always desirable to use new and improved techniques in producing transgenic plants for commercial use. Our technique currently in use will save time, labor, material, with a higher frequency to produce transgenic sorghum plants carrying the target gene, the TLP gene, which provides resistance to stalk rot and tolerance to drought. The new technique is able to be patented.
Publications
- Jeoung, J. M., S. Krishnaveni, S. Muthukrishnan, H. N. Trick, and G. H. Liang. 2002. Optimization of sorghum transformation parameters using genes for green fluorescent protein and ?-glucuronidase as visual markers. J. Hereditas (in press)
- Gao, Z.S., S. Sugita, S. Ikeda, H. W. Cai, T. Sasaki, and G. H. Liang. 2002. Linkage of AFLP markers to lhd 1, a recessive heterohronic gene in Italian ryegrass. Genome 45:752-758.
- Velazhahan, R., J. Jayaraj, J. M. Jeoung, G. H. Liang, S. Muthukrishnan. 2002. Purification and characterization of an antifungal thaumatin-like protein from sorghum (Sorghum bicolor) leaves. J. Plant Diseases and Protection. 109(5): (in press)
- Velazhahan, R., J. Jayaraj, G. H. Liang, and S. Muthukrishnan. 2002. Partial purification and N-terminal amino acid sequencing of a ?-1,3-glucanase from sorghum leaves. Biol. Plant. 45: (in press)
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Progress 01/01/01 to 12/31/01
Outputs
Our experimental data have shown that both microprojectile bombardment and Agrobacterium-mediated transformation are feasible for monocot (wheat, sorghum, and bentgrass) transformation. Using biolistic gun and Agrobacterium strains EHA 105, EHA 101, and LBA 4404, we have been able to transform grain sorghum, wheat, and bentgrass with agronomically useful genes. Green fluorescent protein (GFP) also proves to be a very useful reporter gene in transformation protocols for bentgrass and for wheat. Using GFP as a reporter gene is simple, quick and cost effective (except the initial investment on fluorescent dissecting microscope and the digital camera). It also is non-destructive in contrast to GUS staining. Our unpublished data from bentgrass (cv Crenshaw) and wheat (cv Bobwhite) have shown that Agrobacterium transformation is much more efficient than biolistic gun approach. For bentgrass, the transformation efficiency (number of independent events/total number of calli
co-cultured) is 10-11% and for wheat (number of calli showing GFP 5 wks after inoculation/total number of calli inoculated) is 7-9%.
Impacts
Efficient transformation technique is essential to produce large number of transgenic plants for evaluation. At present, it is biolistic bombardment vs Agrobacterium-mediated transformation, both need tissue culture as a pre-requisite. Because of the efficiency, low copy numbers of the transgene, easy to operate, and cost effectiveness, we will concentrate more on Agrobacterium transformation for crop species. Industries also will adopt quick and reliable protocols for their transformation projects without tedious biochemical testing. Use of GFP reporter gene could provide such efficiency and reliability.
Publications
- Detvisitsakun, C. W. Zhang, S. Muthukrishnan, G. L. Lookhart, and G. H. Liang. 2001. Incorporating a barley Hva1 gene to wheat for drought tolerance. Amer. Soc. Agron. Conf. Abst. 242. Charlotte, NC
- Fu, D. L. 2001. Agrobacterium-mediated transformation of bentgrass with TLP gene. Amer. Soc. Agron. Conf. Abst. 328. Charlotte, NC
- Krishnaveni, J. M. Jeoung, S. Muthukrishnan, and G. H. Liang. 2001. Transgenic sorghum plants constitutively expressing a rice chitinase gene show improved resistance to stalk rot. J. Genet. Breed. 54 : (in press)
- Tuinstra, M., G. H. Liang, C. Hicks, K. D. Kofoid, and R. L. Vanderlip. 2001. Registration of KS 115 Sorghum. Crop Sci. 41:932-933.
- Yu, T. T. 2001. Genetic transformation of bentgrass. Ph.D. thesis. Department of Agronomy. Kansas State Univ. 81 pp.
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Progress 01/01/00 to 12/31/00
Outputs
Sorghum: a rice chitinase gene (chi 11) was incorporated into TX 430 via microprojectile bombardment for resistance to stalk rot caused by Fusarium spp. Overexpression of the gene in the transgenic TX 430 plants showed resistance to the stalk rot while the control was susceptible. There was a correlation between degree of resistance and gene expression as evidenced by western blot. Likewise, the gene encoding thaumatin-like protein or tlp was incorporated into inbred C401 via Agrobacterium tumefaciens (LBA 4404). Some of the transgenic plants not only were resistant to stalk rot but also showed drought tolerance as compared to the control plants. Degree of resistance and tolerance varied among transgenic plants. Bar was used as the selectable marker in both experiments. Bentgrass: using green fluorescence protein (GFP) as a reporter gene, it was shown that transformation via Agrobacterium tumefaciens is feasible. All the regenerated seedlings from transformed calli
also showed the green fluorescence in their leaves, roots, and stems. Wheat: 34 putative plants of Jagger (23) and Lakin (11) were regenerated from microprojectile bombardment with particles coated with Hva1 DNA for drought tolerance. The plants are under analysis for confirmation.
Impacts
Production of transgenic plants with desirable genes for biotic and abiotic resistance is not limited by species incompatibility. Agronomically useful genes from any species can be incorporated into the target crops via either microprojectile bombardment or using Agrobacterium-mediated transformation. The potential is unlimited. With the projected population of 8 billion in 2025, research concerning increase of productivity with using additional land should be explored.
Publications
- Jeoung, J.M. and G.H. Liang. Transgene expression of thaumatin-like protein in grain sorghum. 2000. ASA Abst. Minneapolis, MN p.183.
- Jayaraj, J. and G.H. Liang. Transgenic sorghum plants constitutively overexpressing a rice thaumatin-like protein (TLP) also show elevated levels of beta glucanase. 2000. ASA Abst. Minneapolis, MN p.186
- Sang, Y. and G.H. Liang. Comparative physical mapping of the 18S-5.8S-26S rDNA in three sorghum species. 2000. Genome 43:918-922
- Muthakrishnan,S and G.H. Liang. Pathogenesis-related proteins and their genes in cereals. 2000. Plant Cell, Tissue, and Organ Culture 53:1-23. (Book chapter)
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Progress 01/01/99 to 12/31/99
Outputs
Sorghum, wheat, and bentgrass, all monocots, are being transformed using both biolistic bombardment and Agrobacterium. In addition to existing genes of agronomically importance, we also are making gene construct for B-1,3,-glucanase and testing another selectable marker, Cah, in attempt to replace the bar gene. Green fluorescence protein (GFP) is being used as a reporter gene to replace GUS gene because the former is nondestructive, economic and provide an early detection of the transgenic plants. Sorghum inbreds Tx430 and C401 have been transformed by incorporating G11 and TLP genes. Results were confirmed by Southern and Western analyses. Agrobacterium has been shown to be effective as an alternate transformation tool for bentgrass and the GFP is an alternate reporter gene. The homozygous transgenic sorghum plants in T 2 generation are more resistant to stalk rot caused by Fusarium thapsinum than the control in greenhouse tests.
Impacts
Transgenic plants will play an important role in future. Techniques used to transform cereals need to be compared and refined for better efficiency. Wheat and sorghum, both are monocots, have been transformed using biolistic bombardment, which normally produce only 5-7 transgenics per one thousand pieces of calli.
Publications
- Sang, Y. 1999. Comparative physical mapping of the 45S ribosomal RNA genes in three sorghum species by fluorescence in situ hybridization. M.S. thesis, 31 pp.
- Krishnaveni, S., Liang, G. H., Muthukrishnan, S., and Manickam, A. 1999. Purification and partial characterization of chitinases from sorghum seeds. Plant Sci. 144:1-7.
- Krishnanveni, S., Muthukrishnan, S., Liang, G. H., Wilde, G., and Manickam, A. 1999. Induction of chitinases and B-1,3-glucanases in resistant and susceptible cultivars of sorghum in response to insect attack, fungal infection and wounding. Plant Sci. 144:9-16.
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