GM Sorghum: Applications to Agriculture in Developing Nations

Cindy Lee, Linguistics and Microbial Biology

Katrina Linden, Molecular and Cell Biology

Stephanie Wu, Molecular and Cell Biology

Lab of Peggy G. Lemaux

Agrobacterium-Mediated Transformation of Short-Season and Genome-Sequenced Sorghum

Why Sorghum?

•Biofuel application in the United States

•Staple food crop in much of Africa and the Indian subcontinent

•Despite current use as food, sorghum provides inadequate nutritional value and is difficult to digest

•Related to barley, from which we can use the barley-high-lysine gene (BHL9) to increase nutritional value of sorghum

•Innate ability to grow under harsh conditions

Using Agrobacterium tumefaciens as a vector for stably inserting genes, we are introducing the GFP marker and the selection gene, phosphomannose isomerase (PMI), into the genome of two sorghum varieties. The reduced generation time in N247 speeds transformation success; the genome sequence of BTx623 allows for functional analysis studies. Successful transformation requires a means to culture healthy tissue in vitro from immature embryos, to perform selection for transformed tissues, and finally, to regenerate transformed plants.

From a dietary standpoint, developing nations have the most to gain from productive genetic engineering of a more nutritious, drought-resistant sorghum. From a biofuels standpoint, nations lacking a sustainable source of energy have the most to gain from improving starch digestibility in sorghum. Developing an efficient, reproducible transformation protocol for these two sorghum lines is critical because of the impact it will have on engineering improved varieties of sorghum.

Abstract

Methods

•Competent cells have plasmids engineered with gene of interest and selection factor

•Can inject its plasmid into eukaryotic (plant) tissue and insert gene of interest and markers into plant genome

•Inserts genes into genome in a much more controlled manner than the gene bombardment gun

Protocol Overview

ResultsChoice of cultivars: N247’s shortened growing season accelerates transformation; BTx 623’s fully sequenced genome allows for functional analysis.

•Totipotency: Immature embryos contain undifferentiated cells that can be transformed and later taken back up the developmental ladder.

•Treatment optimization: We manipulate variables such as heat treatment, embryo size, and media composition to maximize transformation frequency and viability.

•Selection markers: GFP allows us to visualize transgenic tissue; phosphomannose isomerase allows us to grow only these tissues.

•Digestion analysis: SDS-PAGE gels, Western blots, and conventional combustion methods characterize protein digestibility ; BLAH for starch.

1. Plant seeds and grow at 28°C and 12 hours of light per day.

2. Collect seeds when embryos are a maximum of 2 mm long.

3. Isolate immature embryos and infect with Agrobacterium after various treatments to optimize transformation. After 3 days co-cultivating embryos and bacteria, plate on antibiotic.

4. Induce callus formation. Choose only white calli, which will give rise to regenerable plant tissue.

5. Identify transgenic tissue by visualizing GFP marker via fluorescent microscopy.

6. Plate transgenic calli on regeneration media to induce root and shoot growth.

7. Transplant regenerated material to soil and cultivate to maturity.

8. Harvest mature panicle of seeds for planting and protein/starch analysis.

9. Use premiere gel-based methods to analyze protein digestibility and BLAH to determine starch digestibility.

Acknowledgments

Many thanks to Peggy G. Lemaux, Joshua Wong, and the Lemaux Lab

ReferencesHowe, A., Sato, S., Dweikat I., Fromm M., Clemente T.; (2006) Rapid and reproducible Agrobacterium-mediated transformation of sorghum; Plant Cell Reports: 25-8: 784-791The Amazing Joshua C WongZhao, Zuo-yu; Cai, Tishu; Tagliani, Laura.; (2000) Agrobacterium-mediated sorghum transformation; Plant Molecular Biology: 44-6: 789-798

Why Agrobacterium?

Variety Treatmentembryos/ treatment

# GFP(+)

% GFP (+)

N247 Heat 43C 3min, 25C 2min 28 23 82

N247 Heat 43C 3min, 25C 2min 28 23 82

N247 No heat treatment 26 19 73

P89 Heat 40C 3min, ice, NC 112 42 37.5

P89 Heat 43C 3min, ice, NC 105 41 39

P89 No heat, NC 116 24 20.7

  Small Medium Large

M2 CI      

M11 CI      

PHI-T CI      

A. Media and Embryo Size

Optimization

B. Heat treatment