PLANT BIOTECH

Modern Plant Biotechnology

Difference from classical plant biotechnology

-Involves rDNA technology

-Involved introduction of foreign genes to create transgenic plants

-Many involve intergeneric transfer of genes

-Known genes are transferred

-Few new genes are transferred

-Multiple inventions came from this

GM crops

Crops can be given extra genes for new and useful characteristics. They are genetically modified (GM)

GM crop useful characteristics in crops

-Pest resistance

-Frost resistance

-Disease resistance

-Herbicide resistance

-Drought resistance

-Longer shelf life

Side note: Balancing traditional/GM foods

How are transgenic plants made?

1. Protoplast fusion

2. Agrobacterium tumefaciens

3. Gene gun

4. Viral

Additional Transgenic factors (how are transgenic plants made)

-Chemical: Calcium Chloride, Liposome

-Physical: Electroporation, Microinjection, Biolistics

-Vectors: Agrobacterium tumefaciens, Tobacco mosaic virus

Protoplast fusion

-When a plant is injured, a mass of cells called a callus may grow over the site of the wound

-Callus cells have the capability to re-differentiate into shoots and roots, and a whole flowering plant can be produced at the site of the injury

-Fortunately the cell wall can be dissolved with the enzyme cellulase, leaving a denuded cell called a protoplast

-The protoplast can be fused with another protoplast from a different species, creating a cell that can grow into a hybrid plant

-Picture shows: dissected leaf→regenerated plant

Agrobacterium tumefaciens

Gram-negative bacterium

Causes crown gall disease

-Virulence genes are on a large tumor inducing, Ti plasmid

-Portion of the Ti plasmid, the T-DNA transfer DNA, is transferred from bacterial cells to plant cells, where it integrates into the plant chromosome

-Picture focuses on: plant infected and healthy plant (tree)

DNA transfer by Agrobacterium tumefaciens

1. Bacterial attachment

2. Sensing plant signals

3. T-DNA and Vir proteins transport

4. T-DNA and effectors nuclear import

5. T-DNA integration and expression

-Agrobacterium: host factors, nucleus, T-DNA (with opine, cytokinin auxin→tumor), VirF, VirF2, T-complex

-Plant cell: plant signals sugar phenolics, acid, vir, ori, T-DNA, opine utilization, opine permease etc

Agrobacterium tumefaciens:

modification of Ti plasmid to enable transfer of GOI

Agrobacterium tumefaciens modification

1.T-DNA tumor causing genes are deleted and replaced with gene of interest driven by plant expressed promoters

2.Genes for DNA transfer and insertion into plant genome are retained (vir genes)

3.Selectable marker gene added to track transformed plant cells. Ex. Hygromycin (antibiotic), Bialaphos (herbicide)

4.Modified Ti plasmid is constructed first in E.coli then transformed into A. tumefaciens

-Picture shows: vir factors encased with circle

Agrobacterium-mediated Transformation

-A. tumefaciens, with GOI, is co-cultured with plant leaf disks with hormone conditions favoring callus development (undifferentiated)

-Antibacterial agents added to kill A. tumefaciens

-Hygromycin or bialaphos added to kill non-transgenic plant cells

-Surviving cells = transgenic plant cells

-Picture shows: Ti plasmid encased in circle inserted to plant

Gene gun

Agrobacterium-mediated transformation

-Ti plasmid carrying desire genes

-Co-cultivation of Agrobacterium with plant tissues

Particle bombardment method

-Metal particles coated with DNA carrying desire genes

-Bombardment of plant tissues with DNA-coated particles

Promoter for plant expression, cloned genes bacterial sequences

Chloroplast Engineering

-Advantages over genome integration:

1. Multiple genes can be expressed as an operon

2. DNA in chloroplast is completely separate from nuclear DNA released in poller -rDNA will not be carried in pollen and transferred to weeds

-Picture shows: Engineered Ti plasmid transfer to plant

Flavr SavR tomato

-Normal tomato→normal mRNA (sense)-->Protein beads→Natural rotting tomato due to PG

-Bacterium: cut out the gene, insert gene

1. Insect resistant gene/tomato plant

2. Cut gene and insert into vector

3. Copy vector and Coat tungsten with DNA vector

4. Load bullet into gene gun

5. Allow vector to enter cells plated on antibiotic media

6. Transfer cell to medium so they can grow

Engineered Resistance to Herbicides

-Roundup = glyphosate

-Systemic herbicide

-Broad spectrum kills monocots and dicots

-MOA: inhibits EPSPS 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase) an enzyme involved in aromatic amino acid biosynthesis

Glyphosate Action on Weeds

Shikimate pathway

-Tryptophan, Phentylaine, Tyrosine

Engineered Resistance to Insects cont

-Texas is the nation's No. 1 cotton producer and 85% of the state's cotton crop is genetically modified (U.S. department of agriculture)

-Problem: there are insects that are now resistant to Cry1Ac

-Solution:

-Picture shows: how it works with Bt proteins in gut/lumen

Engineered Resistance to Insects

Bt crops: protection against insect pests

-Engineered with gene for the toxic Cry (crystal) protein from the bacterium, Bacillus thuringiensis

-Toxin creates holes in the gut wall of insect larvae

Engineered Resistance to Insects Solution

1. Several different Cry genes have been combined to give better protection and overcome resistance

2. Use other insecticidal proteins with completely different mechanism of action from the Cry proteins

Protection against plant viruses

-Papaya ringspot virus (PRSV) reduced papaya production in some Hawaiian islands by as much as 95%

-1998 introduction of transgenic papayas expressing PRSV coat protein saved the industry

PLANT BIOTECHNOLOGY 3

Phytohormones

regulate cellular activities (division, elongation and differentiation), pattern formation, organogenesis, reproduction, sex determination, and resposnses to abiotic and biotic stress

Phytohormones - old timers and newcomers

-Ex: cytokinins, ethylene, salicylates, auxin etc (chemicals)

-Growth

-Seed germination

-Promote flowering

-Promote sex determination in some species

-Promote fruit growth

Genes controlling GA synthesis are important "green revolution" genes

-Tremendous increases in crop yields (the Green Revolution) during the 20th century occurred because of increased use of fertilizer and the introduction of semidwarf varieties of grains

-The semidwarf varieties put more energy into seed production than stem growth, and are sturdier and less likely to fall over

-Distinguished plant breeder and Nobel Laureate Norman Borlaug (1914-2009)

Several of the green revolution genes affect GA biosynthesis

-Semidwarf rice varieties underproduce GA because of a mutation in a the GA20 oxidase biosynthetic gene.

-Picture shows: cytoplasm, active, GA3, vs wild-type, semidwarf

Molecular genetic approaches can limit ethylene synthesis

-Antisense ACC synthase: introduction of antisense constructs to interfere with expression of biosynthesis enzymes in an effective way to control ethylene production