Selection and confirmation of T1 generationmetabolically engineered tobacco for bioproduct

Dilsherpreet Sidhu and Connor GormanBioenvironmental Sciences, Department of Plant Pathology and Microbiology, Texas A&M University

Abstract Results

Introduction and Objectives

Conclusion

Methods

In order to derive homozygous lines from T0 tobacco (N. tabacum) genotypes engineered for squalene production, T1 generation seeds were put through antibiotic selection followed by confirmation of tDNA by gDNA PCR and qPCR expression analysis. The antibiotic concentration used for selection of T1 seed was determined by kill curve analysis using a kanamycin concentration gradient for in vitro germination. Surviving plantlets from selection concentrations killing >25% of T1 seed were transferred to soil and gDNA was isolated using CTAB method. PCR was performed to determine efficacy of selection and confirm tDNA presence. Following acclimation and growth in the greenhouse, RNA was isolated for expression analysis. T1 plant qPCR will be the primary basis of selection for improved genotypes and identifying possible homozygous lines to continue to the T2 generation.

Tobacco (N. tabacum) has been used in recent years as a model crop for metabolic engineering in plants. Currently, it is being used to produce high-value metabolites in the terpene family. Terpenes are a large and diverse class of organic compounds, produced by a variety of plants and lesser so in other natural processes. They may protect the plants that produce them by deterring herbivores and by attracting predators and parasites of herbivores. Terpenes are hydrocarbons, whereas terpenoids contain additional functional groups. The production of these as metabolites in planta however is very low, at levels that would not be deemed a marketable source. Also, terpene metabolism is limited to carbon fixation and downstream metabolism. To prevent downstream metabolism of the desired terpene – squalene – the transgenic approach is to synthesize the compound within the chloroplast. The overall scheme for transgenic squalene biosynthesis involves a synergistic enzyme in carbon fixation (sedoheptulose-1,7-bisphosphatase: SBPase), a carbon redirection from RuBisCo through three enzymes (glycolate-oxidase: GO, catalase: CAT, and malate synthase: MS) into the MEP pathway, leading to two enzymes (farnesyl-pyrophosphate: FPPS, squalene synthase: SQS) that create the squalene compound. Squalene is a natural 30-carbon organic compound originally obtained for commercial purposes primarily from shark liver oil – thus the current method is not sustainable. For this reason funding in production of bioproducts, such as squalene, helps to produce commercial and industrial inputs in an environmentally sustainable fashion.Objectives: •Separate pT8.3&4 lines in to homozygous by selecting them through Kanamycin selection gradient.•Confirm positive plants using gPCR.•Perform qPCR to show elevated transcripts levels.•Use GC-MS to measure squalene.

1. T₁ Seed Selection T1 generation seeds from two high squalene producing lines (pT8.3&4) were germinated in vitro along with their background genotype (G1) and a WT cultivar (1068) using MS media. After cotyledon emergence they were transferred to MS media containing kanamycin concentrations of 0, 50, 75, 100, 150mg*L-1. Three replications were performed to look for ¾ survival, assuming T0 lines are of a single insert. Survival was determined two weeks post treatment based on plant health, where general chlorosis and death were scored as selected, and phenotype similar to control treatment was scored as resistant.

2. gPCR: PCR was performed on the selected pT8.3&4. For the PCR, pT8.3 T₀ was selected as a positive control. Each leaf tissue sample DNA was extracted using the CTAB method. After the genomic DNAs were acquired and checked for quality, PCR were performed using the Econtaq polymerase and gene specific primers for the tDNA SBPase gene. Amplification imaging was conducted using electrophoresis with 1% agarose gel.

3. qPCR: PCR positive lines will under-go expression analysis by qPCR to collect transcription data, primarily for the transgenic SBPase activity.

• The selection of T1 seed varies from selection in leaf disks in regards to antibiotic concentration. Although the selection from the background (G1) was not clear, positive pT8 T1 generation plants were generated and confirmed with PCR. The selection strength, and variation of, may be result of: old stocks, improper storage or thawing, and deactivation by adding it too early to the MS media during preparation.

• qPCR will be done after completion of gPCR results, and plants are not yet of size nor maturity to perform squalene analysis.

• This experience has taught me many useful things such as growing tobacco from seeds, taking care of tobacco, testing certain organisms in selective media for different genes, field work, PCR, and work values which will help me reach my dream of feeding the world.

AcknowledgementsI would like to acknowledge and thank the BESC department and Dr. Joshua Yuan for providing the funds for this opportunity. A big thanks to the Yuan lab specifically Connor Gorman, Zhao Cheng, Ryan Syrenne, Zain Ul Abedin, Mustafa Presswala, Hu Cheng, Dr. Xin Wang, and Dr. Hong Ma for guiding me through this project and taking the time to answer all of my questions. Sponsors for high impact experiences for BESC and the BESC poster symposium include the Department of Plant Pathology and Microbiology,  the College of Agriculture and Life Sciences, the Office of the Provost and Executive Vice President for Academic Affairs. 

1a. Selection Gradient Qualitative Data

1b. Average Kill Curve Data

2. gPCR resultsFigure II. Electrophoresis gel image from gPCR. NEB 1kb ladder (L), pT8 plasmid positive control (5), and H2O negative control (6). SBPase amplicons from the pT8 line samples (1-4).

Figure Ia: Plantlet selection in MS media containing a gradient of kanamycin. Treatments of 0, 50, 75, 100, and 150mg*L-1

kanamycin (increasing left to right) were tested. Photographs in same row are of a single genotype. Selection efficacy evaluated two weeks post transfer to treatments. Survival was determined by phenotype similar to control treatment. Antibiotic sensitive plants exhibited chlorosis and poor root development.

Figure IIb: Kill curve data across the kanamycin treatment gradient of 0, 50, 75, 100, and 150mg*L-1. Lines without kanR+ cassette, G1 (yellow) and 1068 (gray) are standard and treatment controls, respectively. pT8.3&4 (orange) and (blue) respectively. Survival evaluated 2wk post treatment.

L 1 2 3 4 5 6 LL 1 2 3 4 5 6 L