Instructor: John
Finer
214 Williams Hall
OARDC, The Ohio State University
Wooster, OH 44691
Tel: 330-263-3880 (from Columbus, 5-3880)
Fax: 330-263-3887 (from Columbus, 5-3887)
e-mail: Finer.1@osu.edu
Meeting Time: Tuesday and Thursday, 10:00 - 12:00, Williams Hall Conference Room
Office Hours: TBA
Lecture Outline Laboratory Outline
Resources Available: Selected review and journal articles from the following journals: Plant Cell, Tissue, and Organ Culture; Plant Cell Reports; The Plant Cell; Nature Biotechnology; Plant Science; Plant Physiology; Gene; Science; Annual Review of Genetics; Annual Review of Plant Physiology; etc.
Grading: Final grades will be determined from a midterm and final examination, a write-up of a laboratory research project and the laboratory notebook.
Midterm Examination |
30% |
Final Examination |
30% |
Research Project |
30% |
Laboratory Notebook |
10% |
Course Justification: With the current interest in biotechnology and crop improvement, it is beneficial if not essential for students to be aware of what has been done and what can be done with this technology. The potential of biotechnology is great with applications in all areas relating to plant science. This includes agronomy, botany, food science, horticulture, plant pathology, and others. The course is designed to introduce the student to the technical aspects of gene transfer in plants as well as the application of gene transfer technology in basic and applied research. Since most gene transfer systems utilize plant tissue cultures as target tissues, students will first learn some basics of plant tissue culture; including culture initiation and development of regeneration systems via somatic embryogenesis and shoot morphogenesis. DNA construction design and the theory of gene selection will also be covered. A major emphasis will then be placed on the various DNA delivery systems. The final lectures of this course will cover regulatory aspects in gene transfer research; from laboratory guidelines to patent issues to field testing and commercialization of genetically engineered plants.
I Introduction and Overview (1 lecture)
Overview of plant cell transformation. Definitions of terms. Slide presentation of topics which will be covered during the quarter.
II Transformation Target Tissue - Tissue Culture Theory (1 lecture)
Almost all transformation technology is based on tissue culture practices. The theory behind DNA targeting to regeneration competent cells will be presented.
III Plant Regeneration (3 lectures)
In most gene transfer systems, the ability to regenerate transgenic plants from transformed cultures is the limiting factor. Two routes exist for regeneration; shoot morphogenesis and somatic embryogenesis. Some plants regenerate only through shoot morphogenesis, some regenerate only through somatic embryogenesis, some regenerate through both processes, and some do not regenerate. Model plant tissue culture systems exist for both shoot morphogenesis and somatic embryogenesis; tobacco for shoot morphogenesis, carrot for somatic embryogenesis. The theory, principles, and proper interpretation of both model systems will be discussed.
Agronomic plants regenerate primarily through the process of somatic embryogenesis. Regeneration of alfalfa, soybean, and the cereal crops will be given special consideration. The importance of growth regulators, nitrogen source, explant source, cultivar selection, and environmental conditions will be discussed. The influence of these and other factors on the physiology of regeneration will also be addressed.
V DNA Construction Design (2 lectures)
These lectures will cover the molecular genetic considerations of the DNA constructions for transformation. This includes the following:
A. Selectable versus scorable markers
B. Selection of the proper promoter and terminator sequences
C. Targeting specific tissue or organelles
D. Levels of gene expression
E. Antisense constructions
F. Transformation booster sequences
G. Enhancers and intron effects
H. Co or sense-supression
I. Transient versus stable expression
J. Scaffold Attachment Regions
K. Targeted Recombination systems
V Useful Genes (2 lectures)
A large number of genes have been introduced into plants using transformation procedures. Information on the successes and failures as well as potentially useful genes will be presented. Some of these genes include the following:
A. Herbicide resistance
B. Insect resistance
C. Fungus resistance
D. Virus tolerance
E. Seed storage protein modifications
F. Animal virus coat protein
G. Male and female sterility
H. Modification of flower color
I. Phytopharmaceuticals
This list is rapidly growing and new genes will be added to this list periodically.
VI Agrobacterium (2 lectures)
Since Agrobacterium-mediated transformation is an interactive process, both plant and bacterial reactions will be discussed. This includes the following:
A. The wounding process
B. Agrobacterium binding
C. Specifics of target tissue recognition
D. Plant induction of the transfer process
E. The process of DNA transfer
F. Virulence and oncogenicity
G. Tobacco as the model system
H. Use of Agrobacterium mutants
I. Creating the perfect environment for infection
VII Naked DNA systems (3 lectures)
This area is largely technical and the following techniques will be presented and analyzed:
A. Electroporation
B. Particle bombardment
C. Whole tissue electroporation
D. Laser microbeam
E. Silicon carbide fibers
The more uncommon techniques of transformation will be presented in one lecture. The potential and possible advantage of these techniques over others will be discussed. The techniques to be discussed are the following:
A. Pollen transformation
B. Microspore and egg transformation
C. Immature ovule injection
D. Chromosome injection
E. Apex or meristem transformation
F. Macroinjection
VIII. Fate of Introduced DNAs (1 lecture)
Naked DNA transformation systems can give rise to a complex arrangement of introduced DNAs. Agrobacterium gives rise to a simpler and more predictable pattern. Possible mechanisms of DNA integration will be presented. Attempts to better control and take advantage of integrations patterns will also be addressed.
IX. Regulatory and Patent Issues (2 lectures)
The regulations for laboratory research and field trials have not been fully defined and are undergoing constant revision. The following procedures for working with recombinant plants will be presented.
A. OSU Institutional Biosafety Committee applications
B. NIH guidelines
C. APHIS applications for field testing
D. Commercialization applications
There are numerous patents on specific genes, promoters, introns, the process of DNA introduction, the process of plant recovery, and the initial germplasm. Aspects of licensing and technology transfer will be presented.
X. Research Project Report
A research project will be performed by the students in the class. This project will either be a shared class project where the student is responsible for a portion of a large project or an individual student project. All projects must be approved by the instructor. A written report of the research project will account for 30% of the student's grade. The written report will follow the format for a brief scientific manuscript.
Note: All laboratories require observations 1-2 times per week in order to follow response over time. Many laboratories require manipulations at many different time points. The laboratories schedule does not correlate precisely with the topics in lecture as the recovery of stably transformed tissue is a lengthy process. These exercises are performed early in the quarter to allow time for recovery of transgenic tissue.
I Shoot Morphogenesis and Somatic Embryogenesis (1 laboratory)
Students will establish shoot cultures of African violet and streptocarpus. Leaves will be sterilized and various portions of the leaf will be placed on induction medium. Shoot will be transferred to a second medium to induce root formation. Students will transfer embryogenic soybean tissue on an auxin-free medium to induce somatic embryo development.
II Tomato Culture (1 laboratory)
Tomato cotyledons, flowers and leaves will be sterilized and placed on a variety of media containing cytokinin for shoot production and auxin for development of fruit in the absence of fertilization.
III Induction of shoots or somatic embryos (1 laboratory)
Students will be asked to bring in plants of their choice, surface sterilize the tissue and then place it on media containing either 0.5 mg/l NAA with 5 mg/l BA or 0.1 mg/l 2,4-D. The NAA/BA medium is typical for shoot induction while the 2,4-D-containing medium is typical for induction of somatic embryos.
IV Agrobacterium-mediated Stable Transformation of Tobacco (1 laboratory)
Leaf tissue of tobacco will be transformed with genes encoding hygromycin resistance and the Green Fluorescent Protein (GFP). Students will learn the basics of Agrobacterium-mediated transformation and GFP expression.
V Particle Bombardment-mediated Transformation of Soybean (1 laboratory)
Embryogenic cultures of soybean will be bombarded with genes encoding hygromycin resistance and GFP. Students will learn the basics of particle bombardment-mediated transformation and selection for transformed embryogenic material.
VI Evaluation of different constructions for cowpea transformation (2 laboratories)
Various GFP- and GUS-encoding constructions will be introduced into cowpea leaf tissue via particle bombardment. The effect of the different construction components as well as visualization of GUS will be elucidated.
VII Sonication assisted Agrobacterium-mediated transformation of tomato (1 laboratories)
Cotyledonary tissue of cowpea will be subjected to Sonication assisted Agrobacterium-mediated -transformation (SAAT). Tissue will wither be excised or left intact, treated or not treated and analyzed over time for GFP expression The benefits or detrimental effects of sonication will be evaluated.
VIII Gel Electrophoresis (1 laboratory)
The following DNAs will be electrophoresced in an agarose gel: undigested genomic DNA, digested genomic DNA, genomic DNA at the concentration used for PCR, PCR amplified genomic DNA, plasmid DNA, digested plasmid DNA, and marker DNA. The students will learn the basics of DNA electrophoresis. This laboratory will be started before the lecture (gel loaded) and completed afterward the lecture.
IX Unusual DNA introduction methods (1 laboratory)
The students will be exposed to alternate, less efficient methods of transformation. Microlance and silicon carbide whiskers will be evaluated on soybean tissue using gfp detection.
X Protoplast Isolation, Purification and Fusion (1 laboratory)
Protoplasts will be isolated from various tissue sources (flower petal protoplasts provide a nice visual), purified and fused using polyethylene glycol.
