Plant Biotechnology

Plant Tissue Culture Plant cells differ from animals cells in that they are totipotent A totipotent cell is one that can develop into specialized cell types & regenerate an entire organism Tissue culture of plants and the regeneration of complete plants from cells has been done since 1930s This allows large-scale clonal propagation of plants

Plant Cloning

Micropropagation Callus, undifferentiated mass of plant cells Seedlings, each from an individual cell

How Do They Engineer Plants?

Methods of producing transgenic plants

Plant Genetic Engineering Ti plasmid of Agrobacterium tumefaciens A. tumefaciens is a soil microbe that induces crown gall Crown gall is a cancerous mass which forms at the site of infection in plants

T-DNA portion of the Ti plasmid contains genes responsible for the disease T-DNA becomes incorporated into the genome of the plant Part of the T-DNA may be replaced with a foreign gene and used to incorporate this gene into the plants genome A marker is also added to determine which cells have the recombinant gene TIBS 1998, 19:

Biolistics (Biological Ballistics) Useful for engineering corn, rice, wheat, barley, & other crops

Helios Gene Gun Uses an adjustable burst low-pressure helium to sweep DNA- or RNA-coated gold pellets from the inner wall of a small plastic cartridge directly onto a target

Genetically Engineered Plants Herbicide tolerance Insect resistance Crop improvements Functional foods Plants as bioreactors Biofuels Timber improvements Bioremediation

Currently 215 million acres of GM crops grown worldwide Genetically Modified (GM) Crops

>60% of the foods we purchase have GM ingredients 95% of canola is biotech herbicide-tolerant 50% of corn is biotech herbicide-tolerant 35% of corn is biotech insect-resistant 61% of cotton is biotech herbicide-tolerant 52% of cotton is biotech insect-resistant 93% of soybean is biotech herbicide-tolerant (2005 Data, Source :GM Crops: The First 10 Years -- Global Socio-economic and Environmental Impacts; PG Economics Limited ) How Much of What We Eat Is GM?

Who Produces GM Food? BASF Inc. Aventis Cropscience Bayer Cropscience Syngenta Seed Inc. Pioneer Hi-Breed International Inc. Dow Agroscience LLC Monsanto Company

FLAVR SAVR, The First GM Food

The first GM food was the FLAVR SAVR tomato Introduced in 1994 it had delayed ripening characteristics

Fruit softens because polygalacturonase degrades pectin Antisense technology was used to turn off (silence) the polygalacturonase (PG) gene Gene encoding antisense RNA was inserted into tomato cells The antisense RNA finds the normal RNA and hybridizes The cell then degrades this complex, preventing the normal RNA from being translated

PG gene transcription mRNA translation PG gene transcription mRNA Antisense mRNA translation Antisense Technology

Polygalacturonase (PG) is an enzyme that breaks down pectin in ripening fruit walls Plants with an antisense PG transgene produce less PG. Walls soften more slowly Many genes manipulated in the same way to answer basic questions: - what is the role of hormones in ripening? - what do particular enzymes do in fruit walls? Wild-type fruit Antisense PG fruit PG activity Days from 1st colour change Altering Fruit Ripening with Antisense RNA

Most GM tomatoes were used only in canned puree By 1997 FlavrSavr was no longer marketed

Current technologies aid the farmer not the consumer Herbicide Resistance Virus Resistance Insect Resistance

Herbicide Tolerance Methods used to promote crop growth also promote weeds Weeds often outgrow crops and reduce farm output Even though there are about 100 chemical herbicides, weeds still reduce crop productivity by ~12% Problem is that many herbicides kill both crops & weeds This has led to the creation of herbicide tolerant crops

Soybean with no herbicidesSoybean after herbicides Herbicides are used for weed control Herbicide Tolerance Weeds drastically reduce crop yield and quality

Non-selective herbicides (Roundup Ultra and) Roundup® (chemical name: glyphosate) Breaks down quickly in the soil, eliminating residual carry-over problems and reducing environmental impact. Roundup Ready® transgenic varieties of common crops completely resistant to those herbicides

EPSPS Transgene Introduced into Plants Codon usage modified for efficient expression in plants promoter polyA+ Regulatory sequences recognised by plant (either from plant gene or plant virus gene). In this case 35S CaMV promoter Agro. EPSPS tp Transit peptide from plant gene added to allow chloroplast import

Move to greener herbicide Benefits of Glyphosate Tolerance in Crops Can use at any time - can wait until there is a problem Reduced herbicide use Very effective - Weeds very sensitive - GM crop very resistant GM canola surrounded by weeds - glyphosate+ glyphosate

Roundup ® Ready Crops Corn Alfalfa Soybeans Canola Sorghum Cotton Tomato Potato Wheat

1996 Roundup ® Ready Gene Agreement Terms: The farmer must pay a $5 per bag "technology fee The farmer must give Monsanto the right to inspect, monitor and test his/her fields for up to 3 years The farmer must use only Monsanto's brand of the glyphosate herbicide it calls Roundup ®

The farmer must give up his/her right to save and replant the patented seed (replanting seed is a practice as old as agriculture) The farmer must agree not to sell or otherwise supply the seed to "any other person or entity." The farmer must also agree, in writing, to pay Monsanto " times the then applicable fee for the Roundup ® Ready gene, times the number of units of transferred seed, plus reasonable attorney's fees and expenses..." should he violate any portion of the agreement.

Concerns of Roundup ® Ready Crops Spread of resistance genes to weeds Problems with quality of crops e.g.. Cotton bolls falling off prior to harvest Farmers required to purchase seed annually The herbicide is still toxic at high doses

Benefits of Roundup ® Ready Crops Fields no longer need tilling Reduction in weed management costs of up to 37% Decrease in herbicide use by >1lb/acre Overall 74% increase in farmer profits 1 1 U.S. Corn Crop 2003

Roundup ® patent recently expired Researchers have designed new method of resistance Sorted thru 100s of microbes to find a detoxifying enzyme Found 3 genes in Bacillus licheniformis which encode glyphosate N-acetyltransferase (GAT) Using directed evolution generated an enzyme 10000x more efficient ~5yrs to market

There are also varieties of various crops resistant to: Glufosinate Bromoxyil Sulfonylurea

Virus Resistance Yellow Squash resistant to three different viruses were developed by Asgrow Seed Resistance was then transferred to zucchini Virus resistant papaya were developed in the mid 90s This was after a outbreak of papaya ring spot virus destroyed 40% of the Hawaiian crop The varieties called Rainbow ® & SunUp ® are provided free to farmers

Transgenic PRV-resistant papaya has been grown commercially in Hawaii since 1996 Increased virus resistance: Papaya ringspot virus (PRV) Virus has had huge impact on papaya industry in Hawaii - reduction of fresh fruit production directly related to spread of PRV No naturally occurring resistance genes - without GM, papaya industry in Hawaii would be destroyed

promoter Regulatory sequences recognised by plant (either from plant gene or plant virus gene). In this case 35S CaMV promoter and terminator polyA+ PRV coat protein gene expressed from 35S CaMV promoter. Papaya transformed by particle bombardment PRV coat protein gene Confers partial resistance to PRV in one variety (Rainbow) and complete resistance in another (SunUp) Growers have to sign up to careful crop management - minimize virus pressure on transgenics to maintain resistance Papaya Resistant to PRV

Various Cry genes (CryIA(b), CryIA(c), & Cry9C) have been inserted crops such as corn, cotton, potatoes, & rice Pest must ingest a portion of the plant for the toxin to be effective Within hours the gut breaks down and the pest dies Insect Resistance Wt CornBt Corn

What is BT doplnit z prednasky roslinna biotechnologie

Molecular basis of the Bt action

Bt Corn & Monarch Butterflies Cry toxin is expressed in all of the plant as well as pollen Corn pollen can blow onto milkweed growing near corn fields Monarch caterpillars feed exclusively on milkweed An early study showed a possible toxic effect of Bt pollen on monarch caterpillars

Biodiversity / NTO Studies Monarch Butterfly, symbol of nature and wildness in North America. The reports of Bt effects on Monarch butterflies have fueled much emotional debate on the use of biotech crops.

Bt Corn & Allergies Bt corn is approved for human consumption However Starlink ® brand corn is approved only for animal feed Contains Cry9C which may be a potential allergen In 2000 Starlink ® was found in Taco Bell-brand taco shells EPA determined that no one who ate the food was allergic, but they found that it had a moderate potential allergenicity All the products were recalled and Starlink ® corn is not approved for human consumption Starlink ® trademark of Aventis Corp.

Benefits of Bt Corn Crop yield increases by up to 33% 39% less insecticide used Increase in monetary gains by 18% 1 1 U.S. Corn Crop 2003

Concerns associated with GM crops 1.Possible production of allergenic or toxic proteins not native to the crop 2. Adverse effects on non-target organisms, especially pollinators and biological control organisms 3. Loss of biodiversity 4. Genetic pollution (unwanted transfer of genes to other species) 5. Development of pest resistance 6. Global concentration of economic power and food production 7. Lack of "right-to-know" (i.e., a desire for labeling transgenic foods)

How to prevent development of Bt resistance in insects? at least 20% of a farm's corn acreage must be planted to non-BT corn. R = resistant European borer; S = susceptible borer. few Bt-resistant insects surviving in the Bt field would likely mate with susceptible individuals that have matured in the non-Bt refuge. Thus, the resistance alleles would be swamped by the susceptible alleles. < 80%> 20% Strategy will not work if resistance is dominant !!!

Crop Improvements Current research into crop improvements include: Increased growth rate Increased salt tolerance Increased drought resistance Modification of seed oil content

Drought / Salinity Resistance Trehalose is a protectant against many environmental stresses; freezing, osmotic pressure (salinity), heat and dessication. Trehalose (1- -D-glucopyranosyl-glucopyranoside) is synthesised in a two-step process in yeast. Zygosaccharomyces rouxii is one of the most highly osmo-tolerant yeasts – especially to salt Kwon, S.J., Hwang, E.W. & Kwon, H.B. (2004). Genetic engineering of drought resistant potato plants by co-introduction of genes encoding trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase of Zygosaccharomyces rouxii. Korean J. Genet. 26, Transgenic potatoes morphologically identical to parents. trehalose-6-P synthasetrehalose-6-P phosphotase 2ARB LB CaMV 35S nptII NS ter NS prom

Modification of Seed Oil Content

Plant Seed Oils Oils either for cooking or industrial uses are usually extracted from seeds such as, Corn, safflower, sunflower, canola, coconut, flax Seed rely on their stored oil as an energy & carbon sources for germination Most seed oils are made up of unsaturated fatty acids Some tropical oils such as palm & coconut have significant levels of saturated fatty acids

Fatty Acids Triglyceride

Canola Oil -10°C Coconut Oil ° CLard 40.5 ° C 5% Saturated 85% Saturated100% Saturated

Cis-fatty acids, naturally occuring unsaturated acids Trans-fatty acids, artifically generated to keep unsaturated fatty acids from going rancid Trans-fatty acids increase LDL (bad cholesterol) increasing the risk of heart disease

Soybeans with Modified Oil Content Soybeans normally have high amounts of the unsaturated fatty acid linolenic acid Vistive by Monsanto is Roundup Ready soybeans bred to have low amounts of linolenic acid This eliminates the need to hydrogenate the oil

Over 60 million tons of seed oil are used for edible purposes About 15 million tons is employed in industrial usage One of the first plants modified was Rape (Brassica napus oleifera) A member of the mustard family, Rape has been grown for centuries as animal feed and natural lubricant In the 1970s selective breeding led to strains of Rape lacking erucic acid The removal of erucic acid made the oil fit for human consumption, LEAR (low-erucic acid rapeseed)

Rape field full of brillantly yellow flowers

The first transgenic crop with modified oil content was a high lauric oil rapeseed. Rapeseed oil normally contains about 0.1% lauric acid Modified rapeseed contains ~40% lauric acid Lauric acid from GM rapeseed would be used in place of oils from palm or coconut The primary use of lauric acid is in detergents CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -SO 4 - Na + Sodium Lauryl Sulfate a.k.a SDS

Other varieties of GM rapeseed could provide: Steric acid as a substitute for hydrogenated oils Jojoba waxes for use in cosmetics and lubricants Various acids for use in biodiesel Phytases for animal feed Novel peptides for pharmaceuticals Why is rapeseed so versatile? One reason is because it is related to…

Arabidopsis thaliana (Cress) Arabidopsis is a model organism used by scientists to investigate plant development and genomics The Arabidopsis genome was recently completed

Brassica (Mustard) Family

Nutritional Enhancement

Improving Protein Quality Nutritional value of seed storage proteins is often limited - may lack one or more amino acid essential to human health e.g. legume seeds lack cysteine and methionine; other seeds can lack lysine Animals and humans are incapable of making 10 essential amino acids - must obtain in diet Amino acid balance in seeds has been manipulated in laboratory experiments using a number of strategies: - introduce seed storage protein from another species - alter sequence of seed storage protein gene in vitro - manipulate amino acid biosynthetic pathway to increase abundance of particular amino acids Similar strategies have been used to improve protein content and composition in non-seed food crops...

Increased nutritive value of transgenic potato by expressing a nonallergenic seed albumin gene from Amaranthus hypochondriacus Chakraborty et al., PNAS 97, (2000) Potato is the fourth most abundant global crop and used for food, animal feed and production of starch and alcohol Limited in lysine, tyrosine, methionine and cysteine Transformed potato with seed albumin from Amaranthus hypochondriacus which has good amino acid balance p35S CaMV Nos 3 AmA1 pGBSS Nos 3 AmA1 2 alternative constructs. Promoters constitutive or tuber-specific Expression in tuber 5-10 fold higher with GBSS promoter than with 35S promoter pSB8 pSB8G

Changes in protein quality in Amaranthus albumin potatoes 5-8 fold higher essential amino acids in pSB8G transgenics Total protein content also increased (35-45%) Fold increase D E S G H R T A P Y V M C I L F K Amino acid

Golden Rice Inserted genes from other plants & bacteria to produce –carotene Vitamin A deficiencies affect >124 million children worldwide

Vitamin A Vitamin A (retinol) is essential to human growth Our bodies cannot make vitamin A, All carotenoids that contain a -ring can be converted into retinol, and one of the most important carotenoid pro-vitamins is -carotene -carotene is a pigment required for photosynthesis - produced in all plant green tissues

400 million people are at risk of vitamin A deficiency (VAD), particularly in Asia and Africa implicated in up to 2.5 million deaths annually in children under million children go blind each year because of VAD Supplementation programmes have reduced child mortality by up to 50% in target areas supplementation not universal; expensive; misses remote areas VAD makes children especially vulnerable to infections Vitamin A deficiency VAD is most serious in regions where rice is the staple food ; up to 70% children under 5 affected

Vaccine Foods In the early 1990s tomatoes, bananas, & potatoes were proposed as delivery vehicles for vaccines Touted as a simple method of delivering vaccines especially to developing countries Studies have shown plant-produced oral vaccines to increase immunity in mice Potatoes containing Hepatitis B vaccine have been shown to boost immunity in humans

There are concerns about dosing when these crops are directly consumed. Would a dose be? 2 bananas and a tomato What if a person eats too many vaccine potatoes? Also there is concern if the vaccine foods enter the food supply of people who are vaccinated the traditional way

Plants as Bioreactors Plants (crops or cell culture) can be used to produce proteins currently produced by microbes or animal cells The advantage over microbes: The proteins are more like human proteins The advantage over animal cells: Plants cannot become contaminated with mammalian pathogens

Large Scale Biology Corp. (LSBC) uses tobacco plants for drug manufacturing LSBC uses an engineered tobacco mosaic virus (TMV) Recombinant gene is inserted into TMV which infects & replicates in the plants During replication large amounts of the drug are generated The drug accumulates in the leaves which are harvested -galactosidase A purified from tobacco is as effective in treating Fabrys disease as the animal cell derived drug

USDA just approved the use of rice to produce lactoferrin and lysozyme Sigma-Aldrich now sells aprotinin and typsin made in tobacco Duckweed is being used to produce interferon- More and More Plants Are Being Used to Produce Proteins

Plant BioFuels

Cellulosic Ethanol Has higher yield due to the fermentation of sugar released from cellulose Requires the addition of cellulase or acid Agricultural plant wastes (corn stover, cereal straws) Plant wastes from industrial processes (sawdust, paper pulp) Crops grown specifically for fuel production (switchgrass)

BioDiesel 1900 Rudolph Diesel runs his engine on peanut oil Biodiesel is defined as a fuel comprised of mono-alkyl esters of long chain fatty acids derived from vegetable oils or animal fats Transesterification converts triglycerides into methyl esters of fatty acids

U.S. consumes 40 billion gallons of diesel/yr

Timber Biotechnology

Reduction in generation time Trees can take years to flower Those overexpressing the LEAFY (LFY) gene can flower in as little as 7 months. This is of particular value in fruit bearing trees Also allows for rapid analysis mature traits

Alteration in tree size or form Altered expression of a gene involved in hormone synthesis can give wildly differing results Overexpression of GA 20-oxidase gives faster growing trees both in height and diameter and longer wood fibers Inhibition of GA 20-oxidase gives dwarf trees

From left to right: antisense-GA 20-oxidase, wild type, & GA 20-oxidase overexpressing Six Weeks Old Hybrid Aspen

Leaves from GM poplar

Trees engineered to produce lower amounts of lignin These trees grow faster and have greater cellulose content Lignin is a glue-like compound that must be chemically removed from pulp prior to papermaking

Bioremediation

Bioremediation using bacteria has limitations The engineered or specialized bacteria used are often unable to compete with indigenous soil bacteria An alternative is phytoremediation, the use of plants to mop up toxic waste A standard technique for treating soils contaminated with heavy metals (lead or cadmium), or organic pollutants (pesticides) in a cost-effective way