The case of switchgrass C. Neal Stewart, Jr.

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1 Environmental and Regulatory Sustainability of Genetically Engineered Bioenergy Feedstocks The case of switchgrass C. Neal Stewart, Jr.

2 Biomass utilization is a multifactorial problem Land Current cropland Fallow land (CREP, etc) Marginal land Rangeland Forestland Brian Davison, ORNL Feedstocks Grain crops Softwood Switchgrass Animal wastes Agric. residues Bagasse Oil crops Other Process technologies Fermentation & Enzymes Pulping Gasification Thermo/chemical conversion Anaerobic digestion Other Output/Products Chemicals Ethanol Butanol Diesel Wood products Polymers Gas fuels: methane, H2, syngas Ash, fertilizers 2

3 Difference between petroleum and bioenergy feedstocks 3

4 Corn vs. cellulosics Corn Switchgrass 160 bu/acre = 4.5 tons/acre 12 tons/acre 4

5 5 Identify, Understand and Manipulate the Plant Cell Wall Genes Responsible for Recalcitrance

6 Bioenergy and plant genomics: Expanding the nation s renewable energy resources Short rotation hardwoods Today Carbon allocation Tomorrow High yield wood crops Conventional Forestry Yesterday Accelerated Domestication Metabolic Profiling Whole Genome Microarrays 6 Brian Davison ORNL

7 Cell wall structure 7 Nature Reviews Molecular Cell Biology 2, (2001)

8 Switchgrass biotechnology goals Enable high-throughput transformation Tissue culture system Transient expression tools Stable transformation system Vectors for genes of interest Altering cell wall biosynthesis/modified lignin Transgenic plant-expressed cellulases and ligninases Increased yield/domestication Field performance Biosafety/biocontainment SUSTAINABILITY 8

9 Plant cell wall and membrane Plant Cell chromosome T-DNA integrated into plant chromosome T-DNA Ti plasmid Agrobacterium tumefaciens Nucleus 9

10 10

11 Improvement of tissue culture and transformation systems Proliferating callus Agrobacteriummediated transformation 11 After 2 6 weeks place on rooting to root. After 2 weeks place onto regeneration to form shoots. Reduces transformation procedure by 2 months Higher efficiency Pick transgenic callus after two months on selection.

12 What biomass crops where? 12 Lynn Wright et al., ORNL

13 But switchgrass is not the perfect choice Tailored feedstocks for needs Differences in adaptation Resource base Geographic and regulatory considerations 13

14 Stand establishment Lower yields than Misc. 14 Annual Inputs Bad candidate-biotech Disadvantages Vegetative propagation Low genetic variation Agronomy Adaptation-cold Vegetative propagation Inputs

15 Potential biomass of switchgrass Dry Tons of Switchgrass Zero Zero to 300 thousand 15 Up to 1 million Up to 2 million Daniel de la Torre Ugarte

16 Ideal bioenergy feedstock? Widely adapted High yield Low inputs Not recalcitrant to digestion and processing Homogeneous/canalized traits Stress tolerant Farmer friendly Economically friendly Ecologically friendly Or recipe for guaranteeing invasiveness? 16

17 Life is full of choices Platforms Feedstock NEB GJ/ha/yr NER CO 2 Balance Annual Establishment Germpla sm Ag Practice Ecological Benefits Eethanol from starch or sucrose Ethanol from Cellulosic Corn Positive Yes Sugarcane Negative No Sugar beet Positive Yes Sorghum - sweet Positive Yes Miscanthus Negative Yes/No Switchgrass Negative No feedstock Poplar Negative No Biodiesel Soybean Positive Yes Canola Positive Yes Sunflower Positive Yes Yuan et al. Plants to power:bioenergy to fuel the future, Trends in Plant Science, :421 17

18 So, biotechnology could be a bioenergy game changer what about regulations and public acceptance? Biotech food crops still have issues of acceptance and regulations But we don t eat dedicated energy crops Special problems with transgenic perennials Special problems with transgenic plants grown in their geographic center of diversity Gene flow is still a regulatory train wreck 18

19 Biotech tools to mitigate transgene flow: biocontainment Transgenes on chloroplasts Transgenic mitigation: tandem constructs Site specific recombination or zinc finger nucleases Tissue specific apoptosis male sterility Focus on limiting gene flow via pollen 19

20 GM gene deletor Chopping transgenes out of pollen 20

21 21

22 Gene deletor (Luo et al Plant Biotechnol J 5:263) RB RS LAT52 pro Recombinase NOS ter 35S pro GUS-NPTII 35S ter RS LB Pollen genome Pollen-specific promoter LAT52 activates recombinase in polle excision RS LAT52 pro Recombinase NOS ter 35S pro GUS- NPTII35S ter RS Pollen genome RB LB RS 22

23 Gene deletor (Luo et al Plant Biotechnol J 5:263) No recombinase vector Cre-loxP/FRT vector 23

24 Tissue-specific apoptosis Killing pollen cells before they can pollinate 24

25 25 Agroinfiltration a means of rapid assessment of gene expression

26 26 Agroinfiltration marker gene

27 27 Power T via agroinfiltration

28 28 Tissue specific apoptosis

29 Conclusions The choice of feedstock is critical no clear perfect choice, but lots of ways to go wrong Switchgrass will benefit from biotechnology Switchgrass tissue culture system and transformation tools are available Regulatory concerns: gene flow and controlling gene flow are both important Transgenic switchgrass will require biocontainment for deregulation Several biocontainment tools are available We must learn from our past mistakes 29

30 Stewart Lab Thanks also to Yi Li David Ow USDA funding in addition to $ from Sungrant and BESC BESC team Ceres for use of slide 30