Progress 07/01/00 to 07/31/05
Outputs
The project's two main objectives were, (1) to develop an efficient and reproducible protocol for in vitro plant regeneration of 'Jose' tall wheatgrass and (2) to develop a reliable gene transfer technique for 'Jose' using a lignin repressor gene. To achieve them, a wide range of concentrations (0-60um) of thidiazuron (TDZ), 6-benzylaminopurine (BA), kinetin, 2, 4-diphenoxyacetic acid (2, 4-D), picilonic acid (PIC) and naphthaleneacetic acid (NAA) were evaluated for callus and plant formation in mature seeds either alone or in combination in Murashige and Skoog (1962) (MS) or Gresshoff and Doy (GD) (1974) basal medium. Results: The results showed that shoot formation was successfully observed within six weeks of culture. In most cases, callus meristem formed first with subsequent shoot development. Single-and multiple-adventitious shoots were observed irrespective of the medium and growth regulator (GR) treatments. The combination treatments of 20 um kinetin with 2, 4-D
resulted in the highest percentage of shooting callus (88.2%) and (86.0%), and greatest callus shoot averages of 9.2 and 8.2 on MS and GD media, respectively. Shoots were rooted easily and continued normal development. The studies had also developed a reliable Genegun transformation protocol, using GUS gene, which resulted in the transformation of about 90% of blasted calli. Limitation: During the course of these studies, the major difficulty encountered was the lack of availability of 4-coumarate:coenzyme A ligase gene (4CL), which was reported to work on trees (Hu et al., 1999; Nature Biotechnology 17 (8): 750-751) and was the initial target gene in this project. Acting on advice of several experts in the field in effort to find a substitute lignin inhibitor gene, we contacted Dr. Jill Gaskell of Forest Products Laboratory/USDA Forest Service in Madison, Wisconsin, who was very kind and provided us with Lip A and Lip D fungal gene sequences that were associated with lignin
inhibition. As he and other experts kindly warned us, the fungal genes did not express in our crop despite a good gene transfer protocol we had developed. Research development: The activities enabled the development of Langston University (LU) new research programs including (a) Crop Tissue Culture, (b) Crop Transformation, (c) Biotechnology Outreach, (d) Research and Educational Bioinformatics, and (e) Crop Genomics. It had also facilitated the funding of two additional equipment and workshop grants ($315,000), plus one research grant pending ($300,000). Undergraduate research training: At least 18 LU students worked in our lab and honed their lab research skills through the project activities, and some of them had even participated in competitive academic research activities (see poster presentations). Six of them had joined graduate schools include Theophilus Depona (Arizona State Univ.), Shalonda Guy (Univ. of North Carolina), Keith King (Iowa State Univ.), Sheree Booker (Hampton
Univ.), Adrian Sherman (Oklahoma. State Univ.), Joy Ekpo (Oklahoma State Univ.), and Clyde Stearns (Texas. A&M Univ.). Of the remaining, 3 have joined the US workforce and 7 are still in school at LU.
Impacts
Annual winter wheat is the major cool-season livestock forage in large part of the Southern Plains Regions (SPR) of the USA. However, it cannot be consistently grazed throughout the year. 'Jose' tall wheatgrass is potential alternative cool-season perennial forage crop for this region. Thus, should it become widely used by farmers as forage crop, the cost associated with annual land preparation and seeding could be reduced. In addition, it would reduce the competitive usages of wheat to meet food requirements for both animals and humans, and expand the gene pool for sources required to balance animal, thus, human diets. With the reliable regeneration protocol we had developed, abundant clone forage can now be rapidly propagated using tall wheatgrass in a relatively short period of time; and the Genegun transformation technique also developed could be used to introduce any compatible genes of agricultural interest in tall wheatgrass.
Publications
- C Stearns, K Matand, and G Acquaah. 2004. Comparative studies of in vitro regenerated versus in vivo grown plants of 'Jose' tall wheatgrass. Proc. 5th Annual Research Symposium, Langston University, Langston, Oklahoma. Abstr.
- A Washington, N Wu, L Smith, G Acquaah, and K Matand. 2004. The effect of carbenicillin on Agrobacterium-infected callus of tall wheatgrass. Proc. 5th Annual Research Symposium, Langston University, Langston, Oklahoma. Abstr.
- K Matand and G Acquaah. 2003. In vitro plant regeneration of tall wheatgrass. 3rd International Symposium on Molecular Breeding of Forage and Turf. Dallas, Texas. Abstr.
- L Gillard, K Matand and G Acquaah. 2001. Establishment of callus growth curve in tall wheatgrass (Agrotriticum). Proc. Research Day for Regional Universities. P66. University of Central Oklahoma, Edmond, Oklahoma. Abstr.
- G Acquaah, K Matand and A Zipf. 2001. Optimization of parameters for transformation of callus of tall wheatgrass and cassava using a biolistic device. Proc. Research Day for Regional Universities. P57. University of Central Oklahoma, Edmond, Oklahoma. Abstr.
- A Washington. 2004. Tall wheatgrass transformation using Agrobacterium. Proc. Research Day for Regional Universities. University of Central Oklahoma, Edmond, Oklahoma. (Oral presentation).
- K Matand, G Acquaah, B Kindiger, and M Burns. 2005. Organogenesis in tall wheatgrass. Plant growth Regulators Society of America (PGRSA) 33 (2): 76-82.
- K Matand and G Acquaah. 2006. Application of Tissue Culture for Tall Wheatgrass Improvement. In G. Acquaah. Principles of Plant Breeding and Genetics. Blackwell Publ. UK (In press).
- G Acquaah, G. Ude, K Matand, and NJ Tonukari. 2006. Agricultural Biotechnology and Developing Countries: Prospects, Challenges, and Impact. In J. A. Teixeira da Silva (ed), Floriculture, Ornamental and Plant Biotechnology. Global Science Books. Ikenobe, Japan. In press).
- A Mbelem, K Matand, N Wu, G Acquaah. 2005. The assessing of variables that could influence the adoption of biotechnology in middle and high schools within the State of Oklahoma. Proc. Research Day for Regional Universities. University of Central Oklahoma, Edmond, Oklahoma. Abstr.
- Q Relerford, N Wu, L Smith, G Acquaah, and K Matand. 2004. Sensitivity of tall wheatgrass to kanamycin. Proc. Research Day for Regional Universities. University of Central Oklahoma, Edmond, Oklahoma. Abstr.
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Progress 01/01/02 to 12/31/02
Outputs
Genetic engineering is a promising new approach in the improvement of crop plants. Annual winter wheat is the major cool-season livestock forage in a large part of the Southern Plains Region (SPR) of the USA. However, it cannot be consistently grazed through out the year. Because wheat is utilized as a dual-purpose grain and forage crop, the production of grain takes precedence over that forage production. `Jose' tall wheatgrass is a potential alternative cool-season perennial forage crop for this region. It is persistent, and tolerant to flood, salt, and drought. It is a self-seeding perennial, and well adapted to the SPR. The development of a reliable protocol for in vitro plant regeneration is a pre-requisite to in vitro genetic improvement of this crop. In efforts to improve our plant regeneration system, we recently investigated varied concentrations of kinetin, 6-benzylaminopurine (BA), thidiazuron (TDZ), a-naphthaleneacetic acid (NAA), 2,
4-dichlorophenoxyacetic acid (2,4-D), and indoleacetic acid (IAA)used alone or in combination. The results showed that we were able to develop successfully a reliable and reproducible in vitro regeneration protocol of `Jose' tall wheatgrass. The best medium treatment for both callus and plant regeneration consisted of a combination of 20 um of kinetin and 2, 4-D each. Eighty three percent of mature seeds cultured on this medium treatment induced callus with subsequent shoot formation. The next best treatment was the combination of BA and 2, 4-D also at the concentration of 20 um each. The results showed that 2, 4-D used in equimolar combination with either kinetin or BA induced consistently higher callus and shoot formation than either of the growth regulators used alone. Some calli induced only one shoot whereas some others induced multiple shoots. In both cases, all shoots formed roots easily on their respective medium treatments without any further special treatment. Plants were
transferred to the greenhouse where they adapted to the environment with a normal appearance. Our current efforts consist of locating a provider of a suitable lignin inhibitor gene that can successfully reduce the production of lignin in plants. Such gene will be cloned in our lab in an expression vector with a monocot promoter to test its expression level prior to introducing it into `Jose' tall wheatgrass. However, our current limitation consists of not locating a research lab, which has isolated a single lignin inhibitor gene suitable for plant expression. Most lignin inhibitor genes reported are of a fungal origin and have shown little or no plant expression (personal talks with authors). The transformation of some plants with antisense RNA of lignin inhibitor genes at fewer labs where related research is conducted is very low and less reproducible.
Impacts
As stated earlier, annual winter wheat is the major cool-season livestock forage in a large part of the Southern Plains Region (SPR) of the USA. However, it cannot be consistently grazed through out the year. `Jose' tall wheatgrass is a potential alternative cool-season perennial forage crop for this region. Thus, should it become widely used by farmers as forage crop, the cost associated with annual land preparation and seeding could be reduced. In addition, it would reduce the competitive usages of wheat to meet food requirements for both animals and humans, and expand the gene pool for sources required to balance animal, thus, human diets. It would also become a new source of farmers' income.
Publications
- MATAND, K. AND G. ACQUAAH. 2003. Micropropagation of `Jose' tall wheatgrass. Third International Symposium of Molecular Breeding of Forage and Turf (May 18-22)/https://www.register-for.com/mbft/AbstractListings.Asp?Categor y=6. Abstract 57.
- MATAND, K. AND G. ACQUAAH. 2003. In vitro plant regeneration of Tall Wheatgrass. Association of Research Directors/Thirteenth Biennial Research Symposium. March 29-April 2. P 197.
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Progress 01/01/01 to 12/31/01
Outputs
The development of a reliable protocol for in vitro plant regeneration is a pre-requisite to in vitro genetic improvement of 'Jose' tall wheatgrass. In attempt to better our previous results on in vitro regeneration of 'Jose', we adjusted the protocol by broadening the concentration range of growth regulators (0 to 60 um). In addition to thidiazuron (TDZ), 6-benzylaminopurine (BA), and 2,4-diphenoxyacetic acid (2,4-D), growth regulators including picilonic acid (PIC), naphthaleneacetic acid (NAA), and kinetin were also evaluated for callus and plant formation in mature seeds since last May either alone or in combination in Murashige and Skoog medium (MS medium). However, results showed that a low percentage (10%) of cultured seeds induced callus, and that sporadic somatic embryos were observed on calli cultured on a medium treatment where 3um TDZ was combined with 1um 2,4-D. Adjustments made did not improve callus induction and plant formation rates at this time as
expected. Extended transformation studies consisted of optimizing gene gun variables for efficient gene delivery, which included microcarrier size, helium pressure, stopping screen position, rupture disk microcarrier gap, target distance, DNA/bombardment, microcarriers/bombardments, and # of ardments/plate. The monocot GUS constructs used was PA11GUSnDY10s-driven by the maize alcohol dehydrogenase (ash1) promoter and first intron (Ann Blech1, USDA-ARS Plant Gene Expression Center, Albany, CA). The results were consistent with our previous observations, and showed that about 50% of blasted calli was transformed.
Impacts
Annual winter wheat is the major cool-season livestock forage in a large part of the Southern Plains Region (SPR) of the USA. However, it cannot be consistently grazed throughout the year. 'Jose' tall wheatgrass is a potential alternative cool-season perennial forage crop for this region. Thus, should it become widely used by farmers as forage crop, the cost associated with annual land preparation and seeding could be reduced. In addition, it would reduce the competitive usages of wheat to meet food requirements for both animals and humans, and expand the gene pool for sources required to balance animal, thus, human diets.
Publications
- ACQUAAH, A., K. MATAND, and A. ZIPF. 2001. Optimization of parameters for transformation of callus of tall wheatgrass (Agrotriticum) and cassava (Manihot esculenta) using a biolistic device. Proc. Research Day for Regional Universities. p 57. University of Central Oklahoma, Edmond, Oklahoma. Abstr.
- GILLARD, L., K. MATAND, and G. ACQUAAH. 2001. Establishment of Callus growth curve in Tall Wheatgrass (Agrotriticum). Proc. Research Day for Regional Universities. p 66. University of Central Oklahoma, Edmond, Oklahoma. Abstr.
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