AGRICULTURAL BIOTECHNOLOGY

Agricultural BiotechnologyTissue culture and plant breedingTransgenic plants and applicationsAnimal biotechnologyLivestock and breedingCause for concern

AGRICULTURAL BIOTECHAgricultural biotechnology or Green Biotechnology is biotechnology applied to agricultural processes.

Biotechnology SupportsSustainabilityEnhances crop production for sustainable food, feed, fiber and fuel supplies.Promotes resource conservation and energy efficiency.Reduces environmental footprint of agriculture.Improves economic viability for farmers and communities.Advances product safety.

Sustainable AgricultureAn integrated system of plant and animal production practices having a site-specific application that will over the longer term:Satisfy human food and fiber needsEnhance environmental quality and the natural resource base upon which the agriculture economy depends;Make the most efficient use of non-renewable resources and on farm resources and integrate where appropriate, natural biological cycles and controls;Sustain the economic viability of farm operations;Enhance the quality of life for farmers and society as a whole.

Satisfy human food and fiber[and fuel] needs

Increased Yield = More Food Corn: 30 percent increase in yield since 1996 Soybean: 22 percent increase in yield since 1996

Consumer Benefits Fewer saturated fats and no trans-fats Allergen free foods Reduced toxins

Enhance environmental qualityPesticide Applications:Down 500 million pounds

No Till Farming:Up 35 percent

Carbon Dioxide Emissions: Down 10.2 million tons

Efficient Use ofNon-renewable ResourcesWater Availability U.S. cotton farmers saved 93 million gallons of water in last 6 years. More crop per dropFuel 441 million gallons of fuel saved

Economic Viability ofFarm OperationFarm Income: Up $27 billionProduction Costs: Down $1.3 billion

Quality of Life forFarmers and SocietyDevelopment Poverty alleviation Improved health care Educational opportunities Enhanced farmer income translates to viable rural communities

Sustainable BiofuelsAgricultural Biotechnology provides enabling technology for: Yield increases for corn ethanol and soy biodiesel; No till cropping for greater residue collection for cellulosic biomass; Dedicated energy crops

AGRICULTURAL BIOTECHNOLOGYAgricultural biotech can be divided to 2 main areas :i)Plant biotechnologyii)Animal biotechnology

PLANT BIOTECHNOLOGYCan be divided into 2 main areas :Plant Tissue CulturePlant Genetic Engineering

PLANT TISSUE CULTUREBroadly refers to technique of growing plant cells, tissues,organs, seeds or other plant parts in a sterile environment on a nutrient medium.The first commercial use of plant propagation on artificial media was in the germination and growth of orchid plants in the 1920sIt was only after the development of a reliable artificial medium byMurashige & Skoog in 1962 that plant tissue culture really took offcommercially.

Definitionthe culture of plant seeds, organs, explants, tissues, cells, or protoplasts on nutrient media under sterile conditions.

Why do plant tissue culture? Fast commercial propagation of new cultivars Agriculture Fast selection for crop improvement nutritional value,pest control, hardiness Cultivation virus free plants Pharmaceuticals ginseng and taxol Cloning of rare and endangered plants Plant cultures easier and safer to export

HistoryFirst attempted by Haberlandt (1902) - grew palisade cells from leaves of various plants but they did not divide1934 - White generated continuously growing culture ofmeristematic cells of tomato on medium containing salts, yeast extract and sucrose and 3 vit B (pyridoxine,thiamine, nicotinic acid) established the importance ofadditives1953 Miller and Skoog, University of Wisconsin Madison discovered kinetin, a cytokine that plays an active role in organogenesis

Basis for the cell culturePlant cells are totipotentHave the ability to develop into whole plants or plantorgans in vitro when given the correct conditionsNot all plant cells are totipotent. However, there are asufficient number of totipotent cells in the plant .

Basis for Plant Tissue CultureTwo Hormones Affect Plant Differentiation:Auxin: Stimulates Root DevelopmentCytokinin: Stimulates Shoot DevelopmentGenerally, the ratio of these two hormones can determine plant development: Auxin Cytokinin = Root Development Cytokinin Auxin = Shoot DevelopmentAuxin = Cytokinin = Callus Development

Factors Affecting Plant Tissue CultureGrowth MediaMinerals, Growth factors, Carbon sourceEnvironmental FactorsLight, Temperature, PhotoperiodExplant SourceTypesUsually, the younger, less differentiated the explant, the better for tissue cultureGenetics1. Different species show differences in amenability to tissue culture.2. In many cases, different genotypes within a species will have variable responses to tissue culture;

Choice of explantDesirable properties of an explantEasily sterilisableJuvenileResponsive to cultureShoot tipsAxillary budsSeedsHypocotyl (from germinated seed)Leaves

CallusIs a natural response of the plant tissue to woundingA mass of actively dividing undifferentiated cells produced by plant tissue explantCallus can be Resuspended in liquid media to create a susupensionculture of single totipotent cells Or differentiated into plant with the appropriatemanipulations of culture conditions

Steps in Plant Tissue CultureSelection of the explant from a healthy mother plantEstablishment of the explant in a culture mediumMultiplication - the explant gives rise to a callusDifferentiation and organogenesis

What is needed?Explant (some tissues culture better than others)A suitable growth medium Aseptic conditionsGrowth regulatorsFrequent subculturing to ensure adequate nutrition and to avoid the build up of waste metabolites

Choice of explantDesirable properties of an explantEasily sterilisableJuvenileResponsive to cultureShoot tipsAxillary budsSeedsHypocotyl (from germinated seed)Leaves

MediaWhen you make an explant like an axillary bud, you remove it from the sources of many chemicals and have to re-supply these to the explants to allow them to grow.Shoot tip - Auxinsand GibberellinsRoots - water, vitaminsmineral salts and cytokininsLeaves - sugars, GAs

Medium constituentsInorganic salt formulationsSource of carbohydrateVitaminsWaterPlant hormones - auxins, cytokinins, GAsSolidifying agentsUndefined supplements

CarbohydratesPlants in culture usually cannot meet their needs for fixed carbon. Usually added as sucrose at 2-3% w/v.Glucose or a mixture of glucose and fructose is occasionally used.For large scale cultures, cheaper sources of sugars (corn syrup) may be used.

Plant hormones (Growth regulators)Auxins CytokininsGibberellic acidsEthyleneAbscisic AcidPlant Growth Regulator-like compounds

CytokininsAbsolutely essential (no mutants known)Single natural compound, Zeatin. Synthetic analogues Benyzladenine (BA), Kinetin.Stimulate cell division (with auxins).Promotes formation of adventitious shoots.Produced in the root meristem and transported throughout the plant as the Zeatin-riboside in the phloem.

Breeding Applications of Tissue CultureMicropropagationGermplasm preservationSomaclonal variationEmbryo cultureHaploid & dihaploid productionIn vitro hybridization protoplast fusionPlant genetic engineering

Steps of MicropropagationStage 0 Selection & preparation of the mother plantsterilization of the plant tissue takes place

Stage I - Initiation of cultureexplant placed into growth media

Stage II - Multiplicationexplant transferred to shoot media; shoots can be constantly divided

Stage III - Rootingexplant transferred to root media

Stage IV - Transfer to soilexplant returned to soil; hardened off

Through micropropagation, it is now possible to provideclean and uniform planting materials in plantations.Micropropagated plants were found to establishmore quickly, grow more vigorously and taller, have ashorter and more uniform production cycle, and producehigher yields.

Somatic EmbryogenesisThe process of initiation and development of embryos or embryo-like structures from somatic cells The production of embryos from somatic or non-germ cells.Usually involves a callus intermediate stage which can result in variation among seedlingsNot a common micro-propagation technique but is currently being used to produce superior pine seedlings

Somatic EmbryosTissue culture maintains the genetic of the cell or tissue used as an explantTissue culture conditions can be modified to cause to somatic cells to reprogram into a bipolar structureThese bipolar structures behave like a true embryo - called somatic embryos

Plant tissue culture belongs to the lower end of the agricultural biotechnology ladder.Plant tissue culture is a straightforward technique and many developing countries have already mastered it. Its application only requires a sterile workplace, nursery, and green house, and trained manpower. Unfortunately,tissue culture is labor intensive, time consuming, and can be costly.

Why do Plant Tissue Culture?A single explant can be multiplied into severalthousand plants in less than a year this allows fast commercial propagation.Taking an explant does not usually destroy the mother plant, so rare and endangered plants can be cloned safely.Once established, a plant tissue culture line can give a continuous supply of young plants throughout the year.

In plants prone to virus diseases, virus free explants (new meristem tissue is usually virus free) can be cultivated to provide virus free plants Plant tissue banks can be frozen, then regenerated through tissue culture. Plant cultures in approved media are easier to export than are soil-grown plants.

Agricultural biotechnology also uses genetic engineering which is a process of inserting a foreign gene into a plant/animal cell and cloning that cell into a genetically engineered crop/animal. Agricultural Biotechnology:

PLANT GENETIC ENGINEERING

Classical Plant Breeding:Genetic modification following introduction of large amounts of undefined DNA from a genetically similar source.Plant Biotechnology:Genetic modification of a plant cell by introduction of defined DNA(s) from a genetically different source.

Oranges?Strawberries?Potatoes?Apples?Daffodils?Cloning Have you ever eaten/used cloned plants?Turfgrass?Roses?in Plants

CottonPoplarXmas CactusFernSoybeanOhio Buckeye

What is a transgenic?

Transgene the genetically engineered gene added to a speciesTransgenic an organism containing a transgene introduced by technological (not breeding) methods GMOs - Genetically modified organisms GMO - an organism that expresses traits that result from the introduction of foreign DNA

Advantages of Plants forGenetic EngineeringCan reproduce asexually and producelarge numbers offspring.Clone themselvesCan be grown in tissue culture

What kinds of features are desirable to genetically engineer into plants?Herbicide resistanceWhat are herbicides ?Compounds that kills plants!Weed controlInsect resistanceBio PharmingImprovement of nutritional valueDelayed ripening

Agrobacterium Tumefaciens : Naturesplant genetic engineerCauses Crown Gall disease in dicotyledenous plants.Infected plants grow "Galls" or tumors

R. Shanthini 11 March 2010Bt toxin bred GM cropBacillus thuringiensis (Bt) is a common soil bacterium that produces crystals containing proteins that are toxic to certain insects. The Bt gene was successfully inserted into the genome of several crops. The insertion of the Bt gene directly into the genome of crops allowed the crops to constantly produce Bt toxin crystals in all tissues of the plant. No application of any pesticide is required to protect the crop from a large number of pests.

Crop productivity could be increased by introducing such qualities as disease resistance and increased drought tolerance to the crops.

Genes from naturally drought-resistant plants can be used to increase drought tolerance in many crop varieties growing in dry climates so that crops shall use water as efficiently as possible.Source: http://www.ctahr.hawaii.edu/gmo/risks/benefits.aspAdvantages : Agricultural Biotechnology

Increased crop productivity

Advantages : Agricultural Biotechnology

Decreased application of pesticide / herbicide.Perennial production of crops non seasonal dependence.Shorter life cycle for crops.

Agriculture Transgenics On the Market

Insect resistant cotton Bt toxin kills the cotton boll worm

transgene = Bt proteinInsect resistant corn Bt toxin kills the European corn borer

transgene = Bt protein

Virus resistance - papya resistant to papaya ringspot virus transgene = virus coat proteinSource: MonsantoHerbicide resistant crops Now: soybean, corn, canola Coming: sugarbeet, lettuce, strawberry alfalfa, potato, wheat (2005?) transgene = modified EPSP synthase or phosphinothricin-N-acetyltransferase

Biotech chymosin; the enzyme used to curdle milk products transgene = genetically engineered enzyme bST; bovin somatotropin; used to increasemilk production transgene = genetically engineered enzymeSource: Rent Mother NatureSource: Chr. Hansen

Next Generation of Ag Biotech ProductsSource: Minnesota Microscopy SocietyGolden Rice increased Vitamin A content (but not without controversy)transgene = three pathway enzymesSunflower white mold resistancetransgene = oxalate oxidase from wheat

Turfgrass herbicide resistance; slower growing (= reduced mowing)Bio Steel spider silk expressed in goats; used to make soft-body bullet proof vests (Nexia)

First biotech plant product Flavr Savr tomato

Bt Corn Reduces:Insecticide MycotoxinApplication Production

XMonarch larvae on Butterfly weed

Animal Biotechnology

Transgenics are genetically modified organisms with DNA from another source inserted into their genomeA large number of transgenic animals have been createdMice Cows Pigs Sheep Goats Fish Frogs InsectsCurrently, no transgenic animal or animal product is approved by the FDA or USDA for human consumption

Some of the goals of transgenic animal creation are:Research into animal and human diseaseImprove livestock animals Use of animals as bioreactors

Transgenic Animal Creation

Microinjection into the germ line -> transgenic animalGene injected into the male pronuclei

Eggs are infected prior to fertilizationVirus integrates into one of the chromosomesRecombinant Defective Retrovirus

Some of the drawbacks of these methods are:The inserted DNA randomly integrates into the genomeThe eggs must be harvested & fertilized in vitroMore than one copy of the gene may get into the genomeTransgenic Animal Generation

Examples of Transgenic Animals

Transgenic CattleDairy cows carrying extra copies of two types of casein genes produce 13% more milk proteinNot only will this make the milk more nutritious, it would allow for less milk to make more cheeseCurrently the milk from these animals is under FDA reviewThe important difference between this & other transgenics is that the DNA added is not foreign

EnviroPig TMTransgenic pigs express phytase in their salivary glandsPhytic acid in the pig meal is degraded releasing phosphorusThe phosphorus is absorbed by the pigNormally the phytic acid/phosphorus complex passes through the pig and is excreted as wastePig waste is a major pollutant & can cause eutrophication of lakes & streamshttp://www.nature.com/news/2005/050221/images/nbt0305-283-I1.jpg

Transgenic FishTilapiaSalmon/troutCatfishCan grow up to 6 times faster than wildtype fish Most have extra copies of growth hormone (GH) geneTransgenicWildtypehttp://www.nature.com/nbt/journal/v19/n6/images/nbt0601_500a_I1.jpg

Concerns if these supersized transgenic fish got looseTransgenic fish are farm-raised, isolated from wild stocksBut even during farming of wildtype fish, escapes happen frequently (~14 million/yr)What would happen if a large number of transgenic escaped & started breeding with wild fish?http://pewagbiotech.org/buzz/index.php3?IssueID=10

In experiments, transgenic males mated 3x more frequently than the smaller wild malesOffspring of transgenic males lived

http://pubs.acs.org/hotartcl/chemtech/99/jun/fletcher.html+AntifreezewildtransgenicAntifreeze Proteins (AFP)AFPs lower the freezing temperature of blood & fluidsTrout normally do not survive in water below 0.6CTransgenic trout containing an AFP gene & promoter can survive in waters as cold as 1.2C

Animal BioreactorsPharming

http://nolswf.bbc.net.uk/science/genes/gene_safari/pharm/a_pharming.shtml1997, Tracy the sheep, the first transgenic animal to produce a recombinant protein drug in her milkalpha-1-antitrypsin (AAT) treatment for emphysema & cystic fibrosisCreated by PPL Therapeutics & The Roslin Institute

Nexia Biotechnologies transfered the silk gene from Orb spiders into goatsThe resulting male goats were used to sire silk-producing female goatsEach goat produces several grams of silk protein in her milkThe silk is extracted, dried to a white powder, and spun into fibersThe fibers are stronger and more flexible than steelTransgenic male kids carrying silk gene

GTC Biotherapeutics has received approval to sell human anti-thrombin (ATryn) purified from goats milk in EuropeTechnology is not restricted to cows, goats, & sheepThere is interest in using rabbits since housing costs are significantly less & generation time is fasterChickens which produce recombinant drugs in their eggs have been produced by The Roslin Institute

Other Types of Transgenic Animals

Transgene -> Gene coding for a growth hormone

ANDi, the first transgenic primate born in January, 2000224 unfertilized rhesus eggs were infected with a GFP virus~Half of the fertilized eggs grew and divided40 were implanted into twenty surrogate mothersfive males were born,two were stillbornANDi was the only live monkey carrying the GFP(Green Fluorescent Protein ) genehttp://www.ohsu.edu/unparchive/2001/011001andi.shtml

Alba, the EGFP (enhanced GFP) bunnyCreated in 2000 as a transgenic artworkhttp://www.ekac.org/gfpbunny.html#gfpbunnyanchor

http://news.aol.com/story/_a/glowing-pig-passes-genes-to-piglets/20080109143909990001?ncid=NWS00010000000001Transgenic Pigs Pass on the Transgene

GloFish, originally developed in Singapore as a way to monitor water pollution The normally black-and-silver zebrafish was turned green or red by inserting various versions of the GFP geneGlofish are on sale throughout the US except in CaliforniaGlofish retail for about $5 per fish. Normal zebrafish cost around one tenth of the pricehttp://www.nus.edu.sg/corporate/research/gallery/research12.htm

Mouse Knock-out TechnologyGene Targeting

A knockout mouse is a laboratory mouse in which researchers have inactivated, or "knocked out," an existing gene by replacing it or disrupting it with an artificial piece of DNA.The loss of gene activity often causes changes in a mouse's phenotype.

Knock-out technology allows for the specific loss of a gene in miceAllows for the function of the KOd gene to be deduced from the defects seen in the mice can be used to mimick some diseaseUnlike traditional transgenics the trangene is targeted to a specific site in the DNA of the mouse

http://tasq.uq.edu.au/blasto.htmlBlastocyst InjectionBlastocystES cells

http://bunseiserver.pharm.hokudai.ac.jp/gihou/knockout.htmlChimeric mouseThe brown fur comes from ES cells injected into the blastocyst of an albino mouse

Some Examples of Knockout Mice

Many of these knockout mouse models are named after the gene that has been inactivated. For example, the p53 knockout mouse is named after the p53 gene which codes for a protein that normally suppresses the growth of tumors by arresting cell division.

p27 knockout mouse is bigger than the controlThis is not due to obesity, but the skeletal structure is increased in size (everything about the mouse is larger)http://www.bioreg.kyushu-u.ac.jp/saibouE.htmlp27 knockout mouse

http://www.bbc.co.uk/science/genes/gene_safari/wild_west/bigger_and_better02.shtmlGDF8 (Myostatin) knockout mouseMyostatin inhibits muscle differentiation and growth.Over twice the muscle mass of a wildtype mousenormalknockout

Naturally Occurring GDF8 Mutants

FGF5 knockout mouse has long, angora-like hair

Clones and Cloning

Dolly as a lamb with her surrogate motherDolly, First Mammal Cloned From an Adult CellDolly, as an adult

Somatic Cell Nuclear Transfer

What Has Been Cloned So Far?

Somatic Cell Nuclear TransferSheep, Goat, Mouse, Rabbit, Cattle (domestic & wild), Pig, Horse, Mule, Dog, Cat (domestic & wild), Deer

Embryo Splitting (Twinning)Sheep, Cattle, Primate (Rhesus)

Cat CloneDonor Surrogate mother with clone (CC)Out of 87 implants only CC survived to birth

http://www.usatoday.com/news/science/2003-01-21-cloned-cats_x.htmDonor & CloneRainbow & CC

Cloned transgenic cat containing red fluorescent proteinhttp://news.aol.com/story/_a/glowing-pig-passes-genes-to-piglets/20080109143909990001?ncid=NWS00010000000001Transgenic Clones

http://news.nationalgeographic.com/news/2003/05/0529_030529_muleclone.htmlIdaho Gem, first cloned mule1st try 134 implants 2 pregnancies, both failed2nd try 113 implantations 14 pregnancies, one birthSurrogate mother (horse)

In addition to cloning pets or prized livestock, researchers are looking to clone transgenic animalsThis would allow for more uniform expression of transgenic genesNot all transgenic animals express their transgenic genes at equal levelsAlso allows for the rapid expansion to large flocks or herds of transgenic animals

http://www.newrivervalley.com/biotech/day1.htmlPiglets clones created by PPL Therapeutics in 2000The piglets carry a silenced copy of alpha 1,3 galactosyl transferase, or GT, an enzyme involved in organ rejectionIn order to guarantee compatibility a second GT gene must also be silenced

Conservation CloningMany endangered or extinct animals are being cloned or considered for cloningGaurBucardo mountain goat MammothQuaggaBanteng

MammothQuaggaBucardoGaur

Noah, a Banteng clone created by Advanced Cell TechnologiesBanteng are endangered wild bovine from Southeast AsianThis clone was created from frozen tissue of an animal that died in 1980

3 Pig clones, born in 2002, died of heart attacks due to adult clone sudden death syndrome within days of each other by the time they were 6 months old.Dolly had a weight problem, telomeres 20% shorter than normal, she suffered from arthritis, and finally lung cancer due to an infection for which she was finally euthanized at age 6yrs.The success rate ranges from 1 to 3% this contrasts to in vitro fertilization which has a success rate of 50 to 20% Problems with Cloning

http://fig.cox.miami.edu/~cmallery/150/gene/cloning.success.jpg

Nearly all clones show some genetic anomalies Some suffer from placental defects others cardiac defectsMany suffer from large offspring syndrome (LOS)Normal mouse pupCloned mouse pup suffering from LOS

***There a multiple forms of herbicide resistance protect the crop against a variety of herbicides. Glyophosate and glufosinate are the primary classes that the plants are resistant against. Virus resistance is obtained by inserting the viral coat-protein gene into the plant. When this protein is produced in the plant, the viral immune system is activated, and the plant is resistant. *These are bacterial (chymosin) and animal (bST) examples. The chymosin gene is engineered for better cheese production. The bST hormone, engineered and expressed in bacteria, increase milk production in animals. bST was the first major ag biotech product.*Golden rice is enriched for vitamin A. The controversy surrounds the actual utility of the product. Some data suggest the child would have to significantly increase their rice intact of rice for vitamin deficiencies to be alleviated. Because of this knowledge the biotech industry is being accused of promoting a consumer-friendly product only for publicity purposes. The white mold disease is associated with elevated levels of oxalic acid. Resistance is provided by inserting a gene whose product breaks down oxalic acid.*Turfgrass are being targeted because of how much inputs are required for their maintenance. Spider silk protein is one of the strongest known natural products, and a natural for the development of a biologically-based product.