Chapter 18-Genetic Engineering of Plants: Methodology Plant transformation with the Ti plasmid of...

Chapter 18-Genetic Engineering of Plants: Methodology

• Plant transformation with the Ti plasmid of Agrobacterium tumefaciens

• Ti plasmid derived vector systems• Physical methods of transferring genes to plants• Microprojectile bombardment• Use of reporter genes in transformed plant cells• Manipulation of gene expression in plants• Production of marker-free transgenic plants

Why genetically engineer plants?

• To improve the agricultural, horticultural or ornamental value of a crop plant

• To serve as a living bioreactor for the production of economically important proteins or metabolites

• To provide a renewable source of energy• To provide a powerful means for studying the action

of genes (and gene products) during development and other biological processes

Plant transformation with the Ti plasmid of Agrobacterium tumefaciens

• A. tumefaciens is a gram-negative soil bacterium which naturally transforms plant cells, resulting in crown gall (cancer) tumors

• Tumor formation is the result of the transfer, integration and expression of genes on a specific segment of A. tumefaciens plasmid DNA called the T-DNA (transferred DNA)

• The T-DNA resides on a large plasmid called the Ti (tumor inducing) plasmid found in A. tumefaciens

The Ti plasmid of Agrobacterium tumafaciens and the transfer of its T-DNA to the plant nuclear genome

Fig. 18.3 The Ti plasmid of Agrobacterium tumafaciens and its T-DNA region containing eukaryotic genes for auxin,

cytokinin, and opine production.

Fig. 18.1 Infection of a plant with A. tumefaciens and formation of crown galls

Fig. 28-27

Crown Gall on Tobacco

Fig. 17.3 Ti plasmid: structure & function

The infection process:1. Wounded plant cell releases phenolics and nutrients.2. Phenolics and nutrients cause chemotaxic response of A.

tumefaciens3. Attachment of the bacteria to the plant cell.4. Certain phenolics (e.g., acetosyringone, hydroxyacetosyringone)

induce vir gene transcription and allow for T-DNA transfer and integration into plant chromosomal DNA.

5. Transcription and translation of the T-DNA in the plant cell to produce opines (food) and tumors (housing) for the bacteria.

6. The opine permease/catabolism genes on the Ti plasmid allow A. tumefaciens to use opines as a C, H, O, and N source.

Fig. 18.4 The right and left borders of the T-DNA of the Ti plasmid are 25 bp direct “repeats” important for mobilization of the T-DNA by vir gene products

Right 5’-TGNCAGGATATATNNNNNNGTNANN-3’Left 5’-TGGCAGGATATATNNNNNTGTAAAN-3’

Fig. 18.7 The binary Ti plasmid system involves using a small T-DNA plasmid (shown below) and a disarmed (i.e., no T-

DNA) Ti plasmid in A. tumefaciens

Plant genetic engineering with the binary Ti plasmid system

Clone YFG (your favorite gene) orthe target gene in the small T-DNAplasmid in E. coli, isolate the plasmidand use it to transform the disarmedA. tumefaciens as shown.

Transgenicplant

(disarmed)

Table 18.1 Plant cell DNA-delivery methods

Method Comment

Ti plasmid-mediated gene transfer *

Excellent and highly effective, but limited to dicots

Microprojectile bombardment *

Easy and effective; used with a wide range of plants

Viral vectors Not very effective

Direct gene transfer into plant protoplasts

Only certain protoplasts can be regenerated into whole plants

Microinjetion Tedious and slow

Electroporation * Limited to protoplasts that can be regenerated into whole plants

Liposome fusion Limited to protoplasts that can be regenerated into whole plants

* Most commonly used methods

Fig. 18.10 Microprojectile bombardment or biolistic-mediated DNA transfection equipment(a) lab version(b) portable version

When the helium pressure builds to a certain point, the plastic rupture disk bursts, and the released gas accelerates the flying disk* with the DNA-coated gold particles on itslower side. The gold particles pass the stopping screen, which holds back the flying disk, and penetrate the cells of the plant.

*

Table 18.5 Some plant cell reporter and selectable marker gene systems

Enzyme activity Selectable marker

Reporter gene

Neomycin phosphotransferase (kanr) Yes Yes

Hygromycin phosphotransferase (hygr) Yes Yes

Nopaline synthase No Yes

Octopine synthase No Yes

-glucuronidase (GUS) No Yes

Firefly luciferase No Yes

-galactosidase No Yes

Bromoxynil nitrilase Yes No

Green fluorescent protein (GFP) No Yes

Reporter Genes

• For how reporter genes work, see: http://bcs.whfreeman.com/lodish5e/pages/bcs-main.asp?v=category&s=00010&n=15000&i=15010.01&o=|00510|00610|00520|00530|00540|00560|00570|00590|00600|00700|00710|00010|00020|00030|00040|00050|01000|02000|03000|04000|05000|06000|07000|08000|09000|10000|11000|12000|13000|14000|15000|16000|17000|18000|19000|20000|21000|22000|23000|99000|&ns=1322

• GFP Researchers Win Nobel Prize (October 8, 2008)Osamu Shimomura, Martin Chalfie, and Roger Tsien won the Nobel Prize in chemistry

for their work on green flourescent protein, a tool that has become ubiquitous in modern biology as a tag and molecular highlighter, vastly improving our ability to understand what goes on inside cells.

• Perhaps you may even want to see a 10 minute YouTube video on GFP; if so please see http://www.youtube.com/watch?v=Sl2PRHGpYuU

Manipulation of gene expression in plants

• Strong, constitutive promoters (35S Cauliflower mosaic virus promoter or 35S CaMV or 35S)

• Organ and tissue specific promoter (e.g., the leaf-specific promoter for the small subunit of the photosynthetic enzyme ribulosebisphosphate carboxylase or rbc)

• Promoterless reporter gene constructs to find new organ- and tissue-specific promoter (see Fig. 18.15)

• Inducible promoters• Secretion of transgene products by inclusion of a signal peptide

sequence between a root promoter and YFG and growing the transgenic plant hydroponically (YFG product will be secreted)

Fig. 18.26 Marker genes may be a safety issue, so it is best to remove them—here is one strategy

LB RBRecombinase

geneSelectable

marker Target gene

Recombinase recognitionsequence