College of Agriculture & Natural Resources Department of Plant, Soil and Microbial Sciences

Research Expertise, Activities, & Interests

Professor Sticklen's research bridges basic to applied research on addressing the production of industrial biotechnological molecules in cellulosic biomass of feedstock crops for a sustainable biofuel and other biobased economy. She develops and uses bioconfinement methods of genetically engineered feedstock crops to avoid public concerns.

She uses safe methods to develop and use genetic modification (GM) technologies that have no or very minimum concerns among the public. Among these, she has developed corn transgenesis system for production GM corn plants which have low-risk or no risk associated with their pollen flow in the field because the gene products are only produced in leaves and stalks of plants (not in the pollens, seeds or roots).

The specific biobased molecules that she has produced in corn leaves and stalks include: (1) Heterologous biodegradable plastic enzymes, polyhydroxybutyrate, (2) Heterologous microbial endoglucanase (see Figure 1 below), (3) Heterologous microbial exoglucanase (Trichoderma reesei cellubiohydrolase or CBH1), and (4) production of a South African rumen anaerobic microbe cellobiase enzyme in corn biomass. Her team produces all these industrial molecules successfully in crops to replace the same molecules that are at present expensively produced in microbial fomenters.

Her team has produced all of the above molecules in five different plant sub-cellular compartments of corn biomass. These compartments include chloroplast, endoplasmic reticulum, apoplast, vacuole and mitochondria. The reason for multi-targeting of these molecules include; (1) to improve the protein folding and therefore the biological activity of the heterologous proteins because certain of these sub-cellular compartments (for example endoplasmic reticulum) have molecular chaperons capable of assisting and improving protein folding and activity. (2) Also, multi-subcellular targeting of heterologous molecules is expected to increase the level of heterologous molecule production.

Another area of research of Dr. Sticklen is down regulation of lignin biosynthesis pathway enzymes to modify lignin configuration and content. She has used the RNAi technology to down regulate lignin biosynthesis enzymes in corn based on the publicly available mapped corn genome. The down regulation of one major corn lignin biosynthesis pathway enzyme has proven to ease the deconstruction of corn cell walls. The homozygous lignin down regulated corn plants show lower lignin and higher cellulose. Work is in progress to test the digestibility of this corn genotype for feeding of rumen animals. All lignin down-regulated corn plants have developed brown-midrib (Figure 3 below). It is encouraging to see that the lignin down-regulated transgenic corn plants are all morphologically normal (no harms to their vascular systems), have higher cellulose contents, and produce viable seeds.

Research Projects to Improve Profitability of Crops of the U.S.

Bioconfinement of Genetically Engineered Plants:

Safety is an issue for all old or modern technologies, regardless whether it is an auto technology, internet/information technology or biotechnology.  For example, appropriate road signs keep automobiles safely on the pavement and the most sophisticated softwares prevent access to private information transmitted online. Safety of transgenic crops is still lagging while consumer concerns are skyrocketing. The main consumer concern is whether non-transgenic crops could stay away from being contaminated with transgenes. Sticklen's team is performing the following methods to bioconfine genetically modified (GM) crops in the fields.  

1. The flc gene encodes a novel protein that acts as a repressor of flowering. This gene has been cloned and characterized and confirmed causing delay in flowering after transferred to an early-flowering Arabidopsis. Also, when the flc gene was null mutated, plants gave early flowering. When Arabidopsis-expressing the flc gene was verbalized, the level of FLC was decreased and so was the delay in flowering. Also, verbalization, which promotes flowering in the late-flowering Arabidopsis (and in many late-flowering mutants), decreased the level of FLC transcript and protein...  We have transferred this gene to tobacco and corn. So far in tobacco, FLC has caused not only delay in flowering but a significant increase in biomass. We do expect similar results in corn (open pollinated). If so, the delay in flowering of transgenic corn could well allow transgenic maize pollens to mature after all other corn crops in the field have completed their pollination time. As we will to use corn biomass for production of valuable industrial products, increase in biomass is expected to increase accumulation of these products.
2. It has been recorded that chloroplast genome in most flowering plants (including corn) is only maternally inherited. Genetically modified corn has been the center of controversies by consumers because corn is an open pollinated crop and therefore its pollens can easily cross breed with other corn plants in the field. This crop has been mainly transformed through the bombardment of its embryo-derived cells, which contain very small plastids. Although not impossible, this makes the chloroplast transgenesis technology less efficient than if fully developed chloroplasts of totipotent green tissues were bombarded because of the much larger size of the plastids in green cells. Over a decade ago, the Sticklen laboratory developed a system of multimeristem bombardment and efficiently used this system for maize nuclear transgenesis. Other laboratories used this technology for transformation of sorghum, barley and oat. Sticklen's team is working on transferring several beneficially important genes into corn chloroplast genome using the team's multiple shoot bombardment system.

Production of an anti-HIV and anti-arthritis protein in transgenic plants:

The human Secretory Leukocyte Protease Inhibitor (SLPI) is an 11.7 kDa mucosal protein well known for its anti-microbial, ant-viral, and anti-inflammatory activities. At present, one company produces this valuable protein in E.coli and sells the product at about $500/microgram. The high cost associated with the production of SLPI in E. coli is the fact that the non-glycosylated cationic protein expression in E.coli requires extensive denaturation and renaturation processes to refold this molecule into its normal biologically active structure. To solve this problem, successful efforts have been taken to overexpress the human SLPI as a 16.4 KDa "polyhistidine-tagged" protein (HisSLPI) under regulation of baculovirus promoter (ba) in insect cells. The SLPI cDNA is in public domain and therefore we have been able to obtain the baHisSLPI from Dr. Diane Shugars of the University of North Carolina at Chapel Hill, NC.  With collaboration of Dr. E. Crockett of MSU Deprtment of Physiology, Sticklen's team is working to produce the SLPI in its normal biologically active form in plants in a manner that it will not affect plant growth and development. 

Production of a Thermal Biodegradable Plastic in Corn stover (leaf and stem) via genetic engineering:

In the United States, corn is making a marginal market, as it does in most other developed nations.  The U.S. industry has set a goal of achieving 10 percent of all basic chemical building blocks (polymers, enzymes, etc.) from plant-derived renewable sources by the year 2020 which represents at a 5-fold increase in market share from today. Corn, as the major US annual crop with large foliage and fast growth, can play a very important role as a bio-factory for producing these chemicals.

For example, one of our research projects in Sticklen's laboratory is to produce a biodegradable plastic polymer in corn stover (leaves and stems) via genetic engineering of bacterial genes into corn genome.

Petroleum-based plastic products constitute 20 % of our landfills and are notoriously slow to degrade. Although the environmental damage caused by the use of  petroleum-based plastics has been known for many years, these polymers are still widely used.

Production of PHB polymer from bacteria has proven to be very expensive and slow.

PHB-producing bacteria require substrates such as ethanol, sucrose, or glucose that are costly. In bacteria, PHB is produced in dilute aqueous solution, therefore the recovery of PHB from dilute fermentation systems adds to the cost of fermentation as a means of producing PHB. Plants produce carbon sources via photosynthesis in concentrated products. Therefore, the costs of production of PHB in plants may become lower than the costs of its production in bacteria.

The effort to produce starch-based or protein-based biodegradable plastic has its down side of low durability and low plasticity, and may replace certain plastics but not others. Polyhydroxybutyrate and similar polymers have obtained worldwide interest because of their biodegradability in addition to their durability and plasticity. 

Improving Digestibility of corn Silage via Genetic Engineering:

Dr. Sticklen's team members are also working on improving the digestibility of corn silage through genetic engineering. Plant cells are covered with cell walls consisting of lignin and cellulose polymers. Lignin content of silage is normally indigestible and is removed from the animal digestion system almost untouched. However, stomach of animals excretes cellulase enzymes to convert plant cellulose into simple sugars as source of energy. Since approximately 70-80% of plant matter is made of lignocellulose, animals spend a considerable amount of energy to digest the plant cellulase in their feed.   In certain animal feeds, microbial cellulases (endoglucanase and cellobiohydrolase) are added to animal feedstocks to improve digestibility of these polymers in the animal stomach. We are genetically engineering corn plants with the endogluconase gene from Acidothermus cellulolyticu and  cellobiohydrolase gene from Trichoderma reese microorganisms.

Dr. Sticklen laboratory team is expert in plasmid (minigene) construction and genetic engineering of corn (U.S. Patent # 5,320,961, see reference, Patent # 5,281,529 see reference, and Patent # 5,539,095). In fact, her team is the developer of genetic engineering of cereal crops using the Biolistic TM bombardment of meristem primordia (U.S. Patent # 5,539,095). She has successfully transferred different genes into maize using this system.

Production of Biofuel-related enzymes in Corn Stover (Leaf and Stem):

Explained above. 

Improving Turfgrass Species for Resistance to Pathogens, Insects, Herbicides, and tolerance to Drought and Freezing Cold:

Bt Commen bermudagrass: Recently, we developed transgenic common bermudagrass that are resistant to insects (see list of refereed articles above). 

Cloning of a disease resistance gene and its transfer to turfgrass for resistance to brown patch disease:

Chitinases have been implicated in fungal resistance of many plants. Dr. Sticklen's team isolated a cDNA clone ( hs2) encoding a chitinase from a triploid Dutch elm disease resistant American elm (UlmusamericanaNPS-3-487). A plasmid (pKYLX71-pHS2) was constructed using the hs2 sequence under control of cauliflower mosaic virus 35S promoter and NOS terminator. This construct was used to genetically engineer creeping bentgrass (Agrostis palustris Huds.) using Biolistic® PDS-100/He system. A plasmid pJS10 containing the bar herbicide resistance selectable marker gene regulated by the rice actin 1 (Act1) promoter was used as a selectable co-transformation marker. Southern blot analysis of transformants showed that the hs2 transgene copy numbers ranged from 1 to 8. RT-PCR and northern blot hybridization monitored transcription of the transgenes, and western blots determined the hs2 protein expression. Disease resistance profiles of five independent transgenic lines showed two were resistant to Rhizoctonia solani, the causative agent of brown patch disease in greenhouse trials. No correlation was apparent between transgene copy number and the level of protein expression. Copy number and resistance of transgenic plants to Rhizoctonia solani also did not reveal any relationship (See Dr. Sticklen's CV for publication references).

Simultaneous control of weeds and turfgrass pathogens via genetic engineering:

Biolaphos herbicide has been frequently used as selectable agent in plant genetic engineering experiments. We genetically engineered turfgrass with bialaphos resistance gene. We also assessed the antifungal activity of bialaphos and phosphoinothricin (PPT, also known as glufosinate), the active moiety of bialaphos, and tested the simultaneous resistance of plants to herbicide and to pathogenic diseases of turfgrass at the laboratory and at the greenhouse levels. In our studies, bialaphos showed a high level of in-vitro antifungal activity against Rhizoctonia solani (causal agent of brown patch disease) and Sclerotinia homoeocarpa (the casual agent of dollar spot). This herbicide suppressed the mycelial growth of these fungi. Various concentrations of bialaphos were applied to transgenic, bialaphos resistant bentgrass inoculated with fungal pathogens. We reported (see Dr. Sticklen's CV for references) that bialaphos application was able to significantly reduce the symptomatic infection by R. solani and A. homoeocarpa on transgenic plants under control environment. These result indicated that bialaphos can provide a means for the simultaneous control of weeds and fungal pathogens in turf areas with transgenic, bialaphos resistant bentgrass. Work is in progress to field test these transgenic plants for the simultaneous control of weeds and turfgrass pathogens.

Delaying the turfgrass season for sport (golf, football, etc.) fields.

Our hypothesis of our team is that we can develop a long season freezing cold tolerant turfgrass for athletic fields in temperate zones by transferring freeze tolerance genes into turfgrass. Dr. Sticklen's team has successfully engineered turfgrass using several other genes ( bar herbicide resistance, chitinase and the gus reporter) genes regulated by Act1 promoter  (see Dr. Sticklen's CV for references). Work is in progress to transfer three freeze tolerance genes regulated by the rice Act1 promoter to turfgrass genome.

Discovering the functional genes associated with turfgrass diseases.

This is a relatively new research project that involves the use miroarray for discovering transcripts responsible for disease resistance in turfgrass. 

Research Projects to Improve Profitability of Crops of the Developing Nations

Development of corn, wheat, rice, sorghum and millet resistant to insects and pathogens,  and tolerant to environmental extremes (draught, salt,, etc. ) through genetic engineering. Our team has always included scientists from developing nations who wish to get training in these areas/crops. Information about those students and more can be found in the Supervised Students page.