Biotechnology

Plant biotechnology is more than genetic engineering.

• Utilization of plant products and processes

• Tissue culture – growing whole plants from cells

• Gene/Genome Sequencing

• Biofuel production

Henry Ford testing pliability of soybean extract-enhanced car plastic with the back of an axe in early 1947. (Ford Motor Company Archives)

Biotechnology tools have expanded the study of plant physiology

• Targeted manipulation (mutation, over-expression) of genes to help understand their function

• Tissue, cell, and sub-cellular localization of expression products

• Measuring gene expression and/or protein function

Importance of Genome Projects

• Mapping genomes can inform us about the ancestry (common and divergent) of species

• Provides information about what genes are responsible for conferring specific traits

• Used agronomically

  • Breeding using molecular markers based on sequence information can provide better predictability when crossing plants for specific traits

  • Genetically engineering plants based on knowledge about specific sequences

Some sequenced genomes:

• Arabidopsis thaliana (Thale cress, first plant genome sequenced)

• Oryza sativa (Rice, first crop plant sequenced)

• Populus trichocarpa (Poplar tree, first tree sequenced)

• Brassica rapa (Mustard)

• Carica papaya (Papaya)

• Glycine max (Soybean)

• Gossypium hirsutum (Cotton)

• Hordeum vulgare (Barley)

• Zea mays (Corn)

• Vitis vinifera (Wine Grape)

• Triticum aestivum (Wheat)

• Solanum lycopersicum (Tomato)

• Medicago truncatula (Barrel medic, related to alfalfa)

Traditional plant breeding results in the combination of multiple genes in offspring, which results in a lot of random combinations.

Deriving plants using molecular tools has been carried out since ~1983

What are Genetically Engineered Plants?

• Sometimes referred to as “genetically modified”

• Transgenic Plants: plants in which DNA from another species (with which the plant could not cross) has been integrated

• These plants are generated through transformation: the genetic manipulation of a cell resulting from delivery and incorporation of DNA into the recipient genome

• The act of transformation is often referred to as an “event”.

Natural Transformation Processes

• Agrobacterium tumefaciens (a soil bacterium) inserts a fragment of its own DNA into plant cells

  • induces those cells to produce the plant hormones that promote cell division & expansion

  • cells form a tumor-like growth called a crown gall tumor

*Mary Dell Chilton and colleagues recognized the ability of the bacterium to do this, late 1970’s.

Transforming plants using Agrobacterium

The new plant will pass the transgene to its progeny through seed.

Another Transformation Method…

• Agrobacterium effectively transformed dicots, but was not initially effective for monocots and some dicots.

• Biolisitcs or microprojectile bombardment: uses a particle gun or “gene gun”

• DNA of interest is coated on non-reactive metal particles (Gold or Tungsten) and physically forced inside of plant cells at high velocity using gas pressure

Biolistic method – gene gun

After Transformation, Then What?

• Verify that transformation was successful… but how?

  • Analyze DNA (e.g. using PCR) to see if your sequence is there***

  • Select for the trait being engineered

    • If you are engineering for resistance against an herbicide, then apply the herbicide and see if the plant lives!

  • Physical indicators/markers

  • Analyze novel protein production***

On to tissue culture & breeding!

• Take the successfully transformed cells and regrow them into whole plants using tissue culture (the power of parenchyma!).

• Breeding: the new transgenic plants are bred with each other and existing crop plants that are vigorous (hardy and high quality)

• Multiple rounds of breeding ensure that plants stably & consistently express the gene(s) of interest

Partial list of plants that have been transformed

What traits have been engineered into plants?

Input Traits (better yield)

• Insect resistance – Bacillus thuringiensis toxin production

• Herbicide tolerance

• Pathogen resistance (viral, fungal, etc.)

• Drought, cold, and salt tolerance

• Disease Resistance

Output Traits (beneficial/useful qualities)

• Improved nutritional content

• Production of novel compounds

• Reduced oxidation (browning) of cut fruits and other organs

Insect Resistance

• Genes from the Cry gene family of the bacteria Bacillus thuringiensis (Bt), which encode for insecticidal toxin

• Insects ingest protein and eventually starve

• Implementing Bt crops has reduced pesticide application, reduced pest populations, and helped increase potential crop yields

Wild type cotton on left, BT cotton on right Insect Resistance

European corn borer (left) and many others have caused significant damage to corn crops in the U.S.

Insect Resistance

Plants engineered with genes encoding toxin from Bacillus thuringiensis

Herbicide Tolerance

• Weeds are a significant problem in crop fields

• Engineer crop plants with an enzyme that will render them resistant to herbicide effects – Weeds die while engineered plants survive

• Weed resistance to herbicides is real, and a problem (e.g. Palmer Amaranth)

• Multiple strategies developed in response

Adoption of Herbicide Tolerant and Insect Resistant Crops Has Significantly Increased Since Their Introduction in 1996

Virus Resistance

• Credited with helping save the Hawaiian papaya industry against the Papaya Ring Spot Virus

• Rainbow and Sunup - 1999

• Also developed for potato, squash, plum

Chestnut Blight Resistance

• Gene from wheat engineered into American Chestnut that will break down fungal walls

• 

Arctic® Apples – Reduced Browning

Simplot’s InnateTM Potato

• Reduction in brown spot due to harvesting stress and oxidation

• Russett, Burbank Russett, Snowden lines

Freeze Tolerant Eucalyptus for biofuel

• Engineered with cold-responsive gene from Arabidopsis – in the mustard family

• Strategy to increase growth range of Eucalyptus in Southeast

• Temps down to 15°F

Added Nutritional Value

• Example: Golden Rice

• Vitamin A deficiency is a severe problem in some developing areas of the world, leading to blindness, morbidity, and even death

• Beta-carotene is chemically converted to Vitamin A in the small intestine

• Rice engineered with two enzymes in beta-carotene production pathway from daffodil & corn

• Currently grown in the Philippines

Genome Editing Using CRISPR

• Clustered Regularly Interspaced Short Palindromic Repeats - identified in bacteria as a defense against bacterial viruses

• Use specific RNA sequence to guide an enzyme called Cas (CRISPR associated) to target DNA

• Cas enzyme cleaves DNA, which can result in mutations in the target sequence

CRISPR Applications

Example: Waxy corn hybrids generated using CRISPR have modified starch content for industrial applications