Transgenic Plants

A. Why create transgenic plants?

Of purple carnations blue roses....Florigene

B.What genes to transfer?

• One gene to a few genes - the CP4 ESPS example
• Multiple genes - the “Golden Rice” example

C. Constructing transgenes. The minimal requirements for the transgene are a promoter, coding region, and terminator.

General structure: 5’---Promoter …..Coding region…….terminator---3’

Example (Bt gene with nptII selectable marker):

5’ --P35S…Bt…Tnos--//--P35S…nptII…Tnos---3’

Promoter: _________________

• DNA sequence controlling spatial and/or temporal level of transgene expression. Promoters can be
  • constitutive (e.g. CaMV 35S, Ubiquitin, NOS = nopaline synthase)
  • tissue specific (e.g. patatin , which is tuber specific)
  • inducible (e.g. heat shock, photo responsive)

Termination sequence: _______________________

• DNA sequence signaling end of the gene (e.g. T35S, T NOS).

In addition to the target gene, additional elements may be added, such as selectable markers and/or reporter genes

Selectable Markers: _______________________

• Encodes a protein (enzyme) that allows the transformed cells to grow while the growth of the non-transformed cells is inhibited. Examples include
  • Antibiotic resistance (e.g. nptII, hygromycin phosphotransferase)
  • Herbicide resistance (e.g. bar, phosphinothricin acetyl transferase)

Reporter genes: _______________________

• Genes that, upon expression in the transgenic plants, provide a clear indication that genetic transformation did occur, and indicate the location and the level of expression. Examples include.
  • Glucuronidase (GUS) - open source molecular biology tool arising from the discovery of GUS - CAMBIA
  • Luciferase, green fluorescent protein (GFP). An extreme use of GFP

Additional components which may be included in the transgene are enhancers and introns.

D. Transformation procedures: In order for a transgene to be incorporated into the germ line of a plant, it must be incorporated into the genome of a cell which will give rise to tissues which will be asexually propagated or to tissues which will undergo gametogenesis. The two principal mechanisms for transforming tissues with a transgene are the “Gene gun” and the Agrobacterium method.

The gene gun (animation) (also known as micro projectile bombardment or the biolistic method) involves coating small metal particles with the transgene DNA and delivering them into target tissues via an explosive force.

The Agrobacterium method exploits Agrobacterium tumefaciens, a soil bacterium causing a root disease called crown gall. In the case of disease, A. tumefaciens invades the host plant and transfers a piece of its own DNA to the host genome. For transformation, A. tumefaciens has been engineered to carry and transfer transgenes and not to cause disease (Figure a and b).

E. Selection and regeneration:

F. Using the transgenic plants: A plant with one copy of the transgene is termed hemizygote.

G. Current status of transgenic crops:

H. Concerns regarding transgenic plants:

• Cultural/ religious issues - e.g. animal genes in plants
• Dietary concerns - e.g. allergies to novel proteins
• Gene escape - see next section on the creeping bentgrass example
• Non-target organisms - e.g. Bt corn pollen and the Monarch butterfly
• "Wasting precious genes one at a time"
• Ownership
  
  Mendel Biotechnology (Plant transcription factors)
  Monsanto

I. Bringing it all home - Herbicide resistant bentgrass in Oregon

The plants:

Creeping bent grass (Agrostis stolonifera L.)

• Golf greens
• Considered a weed in eight countries

Other Agrostis species (250 worldwide; 34 North American; 24 native).

• Diploid to polyploid
• Sexual propagation
  • Outcrossing
  • A. stolonifera is obligate outcrossing
  • Small seeds
• Asexual propagation
  • stolons
  • rhizomes

The gene:

CP4 ESPS

The case:

2003

• Under APHIS permit, 162 ha test in Jefferson county of glyphosate-tolerant GM creeping bentgrass (event ASR368 by Scotts and Monsanto). The test was conducted within a 4453-ha control area established by the Oregon Dept.. of Agriculture.
  

2004:

• 2.5 ha of production.
• Documented gene flow primarily within ~ 2 km but up to 21 km (Watrud et al. 2004. PNAS 14533-14538)
  • Gene flow documented via seedling tests using
    • protein detection (TraitChek)
    • PCR (using transgenic soybean CP4 ESPS; GenBank Accession No. AF464188.1),
    • sequencing of cloned PCR products.

2005:

• No production
• Continued sampling

2006:

• Detected plants expressing transgene - demonstrated pollen transfer and seed dispersal (Reichman et al. 2006. Mol. Ecology 15: 4243-4255)
• Gene flow documented via using TraitChek, PCR, and sequencing

2007

• Issue settled with civil penalty

Text: 368-370

Other class notes : Power Point notes on genetic engineering of plants from PBIO 427/527

Transgenics and related issues:

Agbios database
Greenpeace - Genetic engineering
Pew Initiative on Food and Biotechnology
Transgenic Crops: an Introduction and Resource Guide
United States Patent and Trademark Office Database

GMO detection (no endorsement - a random sampling!)

• Biogenics
• DNA-TEC
• Genolife
  
• OMIC