In Agricoltura le piante selvatiche sono poco diffuse; lo stesso mais odierno, Tripsacum, dava in origine solo semi per pianta con un guscio duro e somigliava ad un filo d’erba: Pianta non produttiva Pianta poco valida per la nutrizione Oggi il mais porta circa 1000 chicchi a pannocchia e sono molto più digeribili rispetto al passato.

Plant Biotechnology Traditional crossbreeding Recombinant DNA techniques

Agricultural Biotechnology CrossbreedingRecombinant DNA SpeciesRelatedRelated/unrelated Efficiency Susceptibility to external gene influences Genes shuffled in one exepriment 10s of 1000s1 – few Random insertion++ Insecti and herbicides Fertilizers Pollution/run off Potential toxins/risk++

Acri coltivati con piante transgeniche negli Stati Uniti dal 1996 al 2001

How are Transgenic Plants Produced? Agrobacterium tumefaciensAgrobacterium tumefaciens Gene Gun / BiolisticsGene Gun / Biolistics ElectroporationElectroporation Commonly Used Methods:

Duracell DNA containing the gene of interest Plant cell Protoplast Electroporation Technique Power supply DNA inside the plant cell The plant cell with the new gene

A.Tumefaciens gall is not a tiny thing

Ti Plasmid Tumor- producing genes Virulence region Opine catabolism ORI T-DNA region DNA between L and R borders is transferred to plant as ssDNA; T-DNA encoded genes can be substituted by target genes

1. Cytokinins (plant hormone for cell plant division and tumorous growth) 2. Enzymes for indoleacetic acid (auxin) synthesis Another plant hormone (inducing stem and leaf elongation, inducing parthenocarpy and preventing aging) 3. Enzymes for synthesis and release of novel plant metabolites: the opines (uniques amino acid derivatives) the agrocinopines (phosphorylated sugar derivatives). Opines and agrocinopines are NUTRIENTS for A.tumefacies. They can not be used by other bacterial species It provides unique niche for A.tumefaciens Important genes encoded by Ti plasmid Nopaline

Opines are nutrients that are also for quorum sensing The plant cells start to secrete the opines from transferred bacterial T DNA opine diffuses into the surrounding cells and serves as a signal molecules for the conjugation of the agrobacterium (Quorum sensing)

Ti plasmid vector systems are often working as binary vectors Virulence region T DNA region removed ori for A. tum Gene of interest Plant selectable marker Bacterial selectable marker ori for A. tumefaciens ori for E.coli HELPER plasmid Disarmed Ti plasmid DISADVANTAGE: Depending on the orientation, plasmids with two different origins of replication may be unstable in E. coli ADVANTAGE: small vectors are used, which increases transfer efficiency from E. coli to Agrobacterium. No intermolecular recombination is needed

T-DNA vector for plant transformation Left borderRight border T-DNA: Gets inserted into plant chromosome Selectable marker Herbicide/antibiotic resistance Plant-specific Promoter Structural gene (e.g. CRY) Plant-specific Terminator

Design Transgenic Construct pBS backbone Transgene TerminatorPromoter

Design Transgenic Construct Type of promoterNameComment Constitutive35SViral in origin, high level of expression Seed specificOleosinExpression is found in the seed InducibleEthanolActivation of a synthetic promoter

Promoters used for expression in transgenic plants 35S, cauliflower mosaic virus 35S promoter CaMV 35S is a strong promoter that is active in essentially all dicot plant tissues. CaMV is a circular dsDNA genome virus

Procedure for creation a transgenic plant 1. Both plasmids are transfected into A.tumefaciens 2. Plant cell culture is infected with A.tumefaciens 3. Products of Vir genes excised gene of interest within T-DNA and transfer it to plant chromosome PolylinkerKan-resistance geneT-DNA Repeat Gene of interest 4. Plant cells are selected on kanamycine 5. Presence of transgene confirmed by PCR 6. Whole plant could be grown from transformed cells !!!

Callus specialized plant tissues that form over a wound; cork cambium may form and the cells produced will gradually seal the wound Callous cells are easiest cells to transform (as their cell wall is very thin) Protoplasts (no cells wall) is even easier to transfrom, but it is very difficult to grow something viable from it.

Callus nay be produced by manipulation with external concentration of plant hormones Major Plant Hormones Auxins Cytokinins Structurally related to adenine auxin cytokinin  roots auxin = cytokinin  undifferentiated callus Produced by actively growing tissues particularly roots, embryos, and fruits Natural auxin is indoleacetic acid (IAA) Apical meristem is the major site of auxin synthesis

Produce callus  transform callus  stimulate shooting by cytokinin addition Biology of Plants, Raven et. al., Freeman Worth Publishing, cytokinin This procedure is easy for dicotyledone plants (legumes etc)

T-DNA and any DNA contained within it are inserted into a plant chromosome in the transgenic plant and then transmitted in a Mendelian pattern of inheritance.

Monocotyledones are not easy to handle – callus is very difficult to be initiated, and A.tumefaciens is not pathogenic for them 1. Pericarp sholud be pulled back and the immature embryos ( mm) are removed. 2. The immature embryos are placed on a callus induction medium high osmotic media prepare calli for transfomation plantsciences.montana.edu/.../transform1.htm Transformation is performed by gene gun method

DNA with desired gene and antibiotic resistance is coated onto the surface of gold particles. plantsciences.montana.edu/.../transform1.htm vacuum chamber Calli are placed in vacuum chamber, Helium pressure shot DNA into cells Gene gun Coating gold particles with DNA Calli remain on the high osmotic media for 20 hours following shooting.

Each plant is from an independent transformation event Each plant has DNA integrated at a different location Each plant is heterozygous for the introduced DNA Each plant may contain different numbers of insertions

E’ stato osservato che certi bruchi che attaccano alcune piante, muoiono dopo aver ingerito determinati batteri del terreno perchè questi batteri producono una proteina tossica per il bruco. Introducendo il gene del batterio responsabile di questa sostanza tossica nella pianta, si ottiene una pianta transgenica indigesta per i bruchi. ESEMPIO DI PIANTA TRANSGENICA RESISTENTE AGLI INSETTI

Tuberi di patata transgenica resistenti alla Tignola del tubero Patate Transgeniche resistenti alla Tignola del Tubero

Adulto di Dorifora su Pianta di Melanzana Le piante transgeniche di melanzana, cultivar Picentia, che esprimono la tossina bt di Bacillus thuringiensis, sono risultate fenotipicamente normali ed in grado di resistere all'attacco di coleotteri.

Il Virus del Mosaico del Cetriolo è tra i virus più studiati in campo vegetale. Ha un'elevatissima diffusione ed è in grado di infettare più di 700 specie. Nel pomodoro causa serissimi danni alle colture fino alla completa distruzione del raccolto nei casi più gravi. In molte aree agricole, dove il pomodoro era la coltura leader, la sua coltivazione non è più possibile a causa dell'endemica presenza di questo patogeno. Virioni di CMV visti al microscopio elettronico

Pianta di pomodoro Resistente al CMV

Sviluppo partenocarpico può essere ottenuto o selezionando varietà con partenocarpia genetica o spruzzando il fiore in fitormoni auxinici, che inducono l'allegazione del frutto in assenza di fecondazione. I fitormoni auxinici sono prodotti dall'ovulo fecondato e stimolano la crescita del frutto. Pertanto, per costruire piante transgeniche partenocarpiche è necessario introdurre un gene che aumenti il contenuto e/o l'attività dei fitormoni auxinici negli ovuli. Il gene "partenocarpico" deve quindi contenere due tipi di informazione: a) regolativa, che determina non solo dove e quando il gene viene ad essere espresso (i.e. nell'ovulo e nei tessuti derivati dall'ovulo), ma anche il livello di espressione; b) strutturale, che determina il tipo di operazione biochimica da compiere (i.e. aumentare il contenuto e/o l'attività di fitormoni auxinici).

Il pomodoro Flavz Savz sviluppato dalla Calgene non marcisce e non ammuffisce in quanto è stato alterato in esso il gene per l'enzima responsabile della produzione di etilene che induce la maturazione.

Plants synthetize ethylene by themselves (self- regulation of flowering) ACC synthase ACC oxydase

Gene Engineered Eternal Flowers 8 days after ethylene treatement. transgenic non-transgenic 9 days after pollination Non-transgenic transgenic Made by antisense disruption of ACC synthase Antisense gene is expressed in the plant

Fruit ripening and decay (also ethylene dependent) Wild type (left) and antisense ACC oxidase (right) melons harvested 38 days post-pollination, stored at 25ºC for 10 days Fair/S1146.htm

Herbicide Resistance Introduction Phosphoenolpyruvate + Shikimate-3-phospahte 5-Enolpyruvylshikimate-3-phosphate (EPSP) EPSP Synthase Tryptophan

Herbicide Resistance Introduction ESPS synthase mutant –Salmonella typhimurium aroA gene –Pro 101 to Ser 101 mutation –Reduce binding ability to glyphosate

+ Glyphosate X RoundUp Sensitive Plants X X Shikimic acid + Phosphoenol pyruvate 3-Enolpyruvyl shikimic acid-5-phosphate (EPSP) Plant EPSP synthase Aromatic amino acids Without amino acids, plant dies X

Bacterial EPSP synthase Shikimic acid + Phosphoenol pyruvate 3-enolpyruvyl shikimic acid-5-phosphate (EPSP) Aromatic amino acids RoundUp Resistant Plants + Glyphosate With amino acids, plant lives RoundUp has no effect; enzyme is resistant to herbicide

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

Herbicide Resistance Western Blot

Final Test Consumer Acceptance RoundUp Ready Corn Before After

Reduction in herbicide usage with resulting from the use of Roundup Ready soybeans (US). From Doane Market Research, 2000.

Roundup drift is possible, so non-Roundup Ready varieties in the neighborhood may suffer Roundup reduced yield 82 percent; Liberty, 31 percent. Spoon of dirt in the honey barrel (fly in the ointment): Roundup 1/8 driftLiberty 1/8 drift

 -Carotene Pathway in Plants IPP Geranylgeranyl diphosphate Phytoene Lycopene  -carotene (vitamin A precursor) Phytoene synthase Phytoene desaturase Lycopene-beta-cyclase ξ-carotene desaturase Problem: Rice lacks these enzymes Normal Vitamin A “Deficient” Rice

The Golden Rice Solution IPP Geranylgeranyl diphosphate Phytoene Lycopene  -carotene (vitamin A precursor) Phytoene synthase Phytoene desaturase Lycopene-beta-cyclase ξ-carotene desaturase Daffodil gene Single bacterial gene; performs both functions Daffodil gene  -Carotene Pathway Genes Added Vitamin A Pathway is complete and functional Golden Rice

BetaSweet® carrot Contains approximately 50% more Beta Carotene than normal carrot Beta carotene is a potent cancer-fighting antioxidant. dark maroon-purple color (as also anthocyanine (another antioxidant) is added) taste similar to regular carrots, but have a very crispy texture, which is easier to chew Produced by Texas A&M University.

Use of Rice to prevent and treat vitamin A and iron deficiencies Iron deficiency is the most common nutritional disorder Iron-enriched transgenic rice: 1 gene increases Fe content ( ferritin) 2 genes increase Fe absorption ( phytate, cysteine) DOUBLE TRANSGENIC RICE b-carotene-enriched rice crossed with iron-enriched rice; b-carotene enhances iron uptake Free distribution to farmers in developing world

Metabolic Pathways are Complex and Interrelated Understanding pathways is critical to developing new products

Modifying Pathway Components Can Produce New Products Modified Lipids = New Industrial Oils Turn On Vitamin Genes = Relieve Deficiency Increase amino acids = Improved Nutrition

MOLECULAR F(PH)ARMING “The use of plants as bioreactors for the production of recombinant proteins” A wide range of proteins produced in plants to date ranging from pharmaceuticals to commodity enzymes

Currently most commodity enzymes are produced in capital intensive fermentation systems

An alternative production system is to use transgenic plants and capture solar energy

Fields of Transgenic Alfalfa Will Replace Fermentation Systems

Spider silk - many times stronger than Kevlar!

Applications of spider silk  bullet-proof clothing  wear-resistant light weight clothing  ropes, nets, seat-belts, parachutes  biodegradable bottles  bandages, surgical sutures  artificial tendons  supports for weak blood vessels

Milk Silk!

Methylmercury bioconcentrates in fish six to seven orders of magnitude above concentrations found in polluted waters and constitutes 90–100% of their total mercury content. Mercurium Serious environmental threat as it enter chains Ionic mercuruim, easily eliminated Scott P. Bizily et al., 2000 BIOMAGNIFICATION

The problem? Methylmercury is highly toxic!

Organomercurial lyase (merB) Mercuric Reductase (merA) Mercury Resistance Bizily et al, 2000

Hypothesis: If plants are genetically engineered with the bacterial mercury resistance genes (merB and merA), then they should be able to convert the very toxic methylmercury (CH 3 Hg + ) to the less toxic, volatile elemental mercury (Hg 0 ).

mer B, merA/merB-1, mer A, and wild-type plants Bizily et al, 2000 (Figure 2A-D) A: 0 ppm CH3HgCl B: 0.2 ppm CH3HgCl C: 1 ppm CH3HgCl D: 2 ppm CH3HgCl

Vaccine production in edible plants

Inexpensive oral vaccines with no refrigeration 500 million vaccines are administered each year in developing countries Needles and syringes can be deadly themselves (AIDS) –Needles and syringes are often refused –They are not properly sterilized

Each Plant has pros and cons Potatoes: good because can be stored for long periods without being refrigerated Disadv.: needs to be cooked in order to eat and heat can denature proteins Most beneficial because some potatoes are eaten raw in some countries and heat does not completely destroy protein

Each plant has pros and cons Bananas: no cooking required and grow in most countries Disadv.: banana trees take years to mature and spoils rapidly Tomatoes: grow fast Disadv.: spoil rapidly

Other foods Lettuce Carrots Peanuts Rice Wheat Corn soybeans

HBsAg – major antigen of hepB virus HBs Ag alone is sufficient to mount immune response Total immunization is a preventive answer

Transgenic lupin (Lupinus luteus L.) and lettuce (Lactuca sativa L.) with hepatitis HBs antigen Kapusta, J. et al., FASEB J Anti-HbsAg antibodies titre in Mice fed with transgenic lupin callus 5 g in 1 dose Anti-HbsAg antibodies titre in Mice fed with transgenic lupin callus 1 g each of the 5 days

Three human volunteers are fed with transgenic lettuce 200 g 1 st dose, 150 g 2 nd dose

Hepatitis B vaccine in Banana Hepatitis B vaccine now costs $100 to $200 a dose Vaccine banana would cost only a few cents per dose. Just 24 acres of land could produce enough bananas to vaccinate all Mexican children under the age of 5.

Norwalk virus (calicivirus) acute diarrhea and vomiting (2-3 days gastroenteritis), abdominal cramps, myalgias, malaise, headache, nausea, and low-grade fever 50% the outbreaks of acute infectious nonbacterial gastroenteritis in the United States the second most common cause of illness in American families (after common cold) No treatment available CDC data: 23 million US people infected by Norfolk annually. 1.4 million cases by salmonella. 79,000 by E. coli contamination 2,500 cases by listeriosis

Capsid-based plant-derived vaccine for Norwalk virus Serum antibody responses of mice fed with Norwalk potato (4g) CT – inert component of cholera toxin (immunoboosting agent) Hugh S. Mason et al, 1998

Human Trial of Norwalk vaccine Tacket et al., The Journal of Infectious Diseases 2000;182: g of raw, peeled, diced potato ( mkg of NVCP coat protein). 24 volonteers (20 exp + 4 controls) 19 out of 20 start to produce specific IgA antibody- secreting cells. 4 out of 20 start to produce specific IgG antibodies Titer of serum IgG anti-NVCP: 1 : 67 before immunization 1 : 757 after immunization among responders 1 : 62,414 after real infection (fade after 2 years) Side effects: nausea in 20% of all volonteers (raw potato)

I rischi delle Biotecnologie L’agricoltura è di per se una cosa non naturale perché comporta: Distruzione di foreste; Modifica dell’ambiente; Riduzione della biodiversità; Inquinamento ambientale. Nessuna tecnologia è esente da rischi e da problemi, la domanda da porsi é: Il rischio vale la candela ? Ovvero i benefici ottenuti superano i rischi ? Per l’agricoltura convenzionale la risposta sinora è stata SI. Le piante transgeniche comunque NON possono essere innocue del tutto.

Quali rischi sono possibili: Effetti tossici sull’uomo; Danni all’ambiente; Inutilità per i paesi ricchi; Gestione commerciale; Incapacità di risolvere la fame nel mondo Gli OGM scatenano allergie: Il 2-4% dei bambini e l’1-2 degli adulti è allergico a soia, latte, farina, riso, arachidi, crostacei etc Le legislazioni dei paesi produttori salvaguardano ciò evitando la vendita di prodotti che possono contenere proteine “allergeniche” se l’organismo di partenza è allergenico.

Altro rischio paventato è di creare resistenze indesiderate agli antibiotici es kanamicina, neomicina etc. Gli antibiotici servono per “trovare” tra le tante cellule quelle trasformate, diventate resistenti agli antibiotici, mentre le non-OGM sono sensibili e periscono. Il rischio è il passaggio di tale resistenza ai nostri batteri intestinali.

Ciò è altamente improbabile; nel nostro intestino ci sono miliardi di batteri che mutano continuamente per cui le resistenze agli antibiotici sono sempre presenti, ma manca la spinta selettiva ovvero non ingeriamo gli antibiotici usati per selezionare gli OGM. Nel nostro DNA NON ci sono geni di vacca oppure di mele eppure ingeriamo tutti i giorni carne e frutta. Il 50% degli antibiotici è usato in zootecnia e con carni crude e/o insaccati convogliamo molti batteri resistenti agli antibiotici e selezionati dagli animali.

Un quesito è se possono le piante OGM involontariamente trasmettere per riproduzione sessuata ad altre piante il loro gene acquisito ? Usare piante maschio sterili; Non consentire coltivazioni non-OGM vicino ad OGM; SI SI può evitare facendo: -Integrare il gene nel DNA del cloroplasto (molte piante trasmettonoi cloroplasti per via materna per cui il polline non è OGM);

Gli OGM non riducono la biodiversità. A ridurla è stato l’uomo per aspetti commerciali; le specie più produttive sono favorite per il mercato e oggi ci sono solo 3-4 specie di mele rispetto alle 200 varietà di un secolo fa.

« Junk » DNA (1) nei mammiferi i geni rappresentano il 2-5% del DNA totale sequenze regolatorie e introni rappresentano un altro 2% Qual’e’ la funzione del restante %? Il ruolo di questo DNA non e’ stato scoperto ma fino a pochissimo tempo fa si pensava che non avesse funzioni. questo 93-95% e’ percio’ chiamato « junk DNA ». Il « Junk DNA » non codifica per nessuna proteina.

«Junk » DNA (2) Organismi complessi hanno piu’ geni di organismi semplici La proporzione di geni sul DNA totale e’ piu’ bassa in organismi complessi

« Junk » DNA (3) Comunque non ci sono prove che il « junk DNA » non ha funzioni. Anzi la complessita’ del sistema di regolazione genica degli organismi superiori potrebbe lasciar supporre un qualche ruolo.

« Junk » DNA (4) Il fatto che il « junk DNA » possa avere un qualche ruolo e’ fondamentale per l’ingegneria genetica e per la piante transgeniche in particolare. Infatti nel produrre una pianta transgenica si assume che l’inserzione di DNA in una regione di « junk DNA » non causi altre alterazioni nelle funzioni della pianta. Se questa assunzione non e’ piu’ vera significa che ci possiamo aspettare alterazioni imprevedibili.