In Agricoltura le piante selvatiche sono poco diffuse

In Agricoltura le piante selvatiche sono poco diffuse; lo stesso mais odierno, Tripsacum, dava in origine solo 10 -20 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 http: //www. insp. mx/xcongreso/ponencias/5

www. quirkyworks. com/design/ Samples/plant-breeding. jpg

Agricultural Biotechnology Species Crossbreeding Recombinant DNA Related/unrelated 10 s of 1000 s 1 – few + + Efficiency Susceptibility to external gene influences Genes shuffled in one exepriment Random insertion Insecti and herbicides Fertilizers Pollution/run off Potential toxins/risk http: //www. insp. mx/xcongreso/ponencias/5

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

How are Transgenic Plants Produced? Commonly Used Methods: • Agrobacterium tumefaciens • Gene Gun / Biolistics • Electroporation

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

A. Tumefaciens gall is not a tiny thing

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

Important genes encoded by Ti plasmid 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). Nopaline Opines and agrocinopines are NUTRIENTS for A. tumefacies. They can not be used by other bacterial species It provides unique niche for A. tumefaciens

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 T DNA region removed Gene of interest Plant selectable marker Bacterial selectable marker Disarmed Ti plasmid ori for E. coli Virulence region ori for A. tumefaciens HELPER plasmid ori for A. tum 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 Plant-specific Promoter Structural gene (e. g. CRY) Selectable marker Herbicide/antibiotic resistance Plant-specific Terminator Left border Right border T-DNA: Gets inserted into plant chromosome

Design Transgenic Construct Promoter Transgene Terminator p. BS backbone

Design Transgenic Construct Type of promoter Name Comment Constitutive 35 S Viral in origin, high level of expression Seed specific Oleosin Expression is found in the seed Inducible Ethanol Activation of a synthetic promoter

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

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 T-DNA Repeat Polylinker Kan-resistance gene T-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 Natural auxin is indoleacetic acid (IAA) Apical meristem is the major site of auxin synthesis Cytokinins Structurally related to adenine Produced by actively growing tissues particularly roots, embryos, and fruits auxin < CYTOKININ shoots AUXIN > cytokinin roots auxin = cytokinin undifferentiated callus

Produce callus transform callus stimulate shooting by cytokinin addition + cytokinin This procedure is easy for dicotyledone plants (legumes etc) Biology of Plants, Raven et. al. , Freeman Worth Publishing, 1999

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 (0. 5 - 1. 0 mm) are removed. 2. The immature embryos are placed on a callus induction medium Transformation is performed by gene gun method high osmotic media prepare calli for transfomation plantsciences. montana. edu/. . . /transform 1. htm

DNA with desired gene and antibiotic resistance is coated onto the surface of gold particles. Calli are placed in vacuum chamber, Helium pressure shot DNA into cells Gene gun Coating gold particles with DNA vacuum chamber Calli remain on the high osmotic media for 20 hours following shooting. plantsciences. montana. edu/. . . /transform 1. htm

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

Each plant is from an independent transformation event TF #2 TF #1 insertion in chrom 2 insertion in chrom 1 insertions in chrom 2 & 3 TF #3 • Insertions at different locations • Each plant is heterozygous for insertion • Plants may have multiple insertions

ESEMPIO DI PIANTA TRANSGENICA RESISTENTE AGLI INSETTI 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.

Patate. Tuberi di patata transgenica resistenti alla Tignola del tubero 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.

Virioni di CMV visti al microscopio elettronico 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.

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 (selfregulation of flowering) ACC synthase ACC oxydase

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

Fruit ripening and decay (also ethylene dependent) www. nf-2000. org/secure/ Fair/S 1146. htm Wild type (left) and antisense ACC oxidase (right) melons harvested 38 days post-pollination, stored at 25ºC for 10 days

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

Herbicide Resistance Introduction • ESPS synthase mutant – Salmonella typhimurium aro. A gene – Pro 101 to Ser 101 mutation – Reduce binding ability to glyphosate

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

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

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

Herbicide Resistance Western Blot

Final Test Consumer Acceptance Round. Up Ready Corn Before After

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

Spoon of dirt in the honey barrel (fly in the ointment): Roundup drift is possible, so non-Roundup Ready varieties in the neighborhood may suffer Roundup 1/8 drift Liberty 1/8 drift Roundup reduced yield 82 percent; Liberty, 31 percent. http: //www. lsuagcenter. com/Communications/Louisiana. Agriculture/agmag/images/43_3/crop_response 3. jpg

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

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

Beta. Sweet® 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 Turn On Vitamin Genes = Relieve Deficiency Modified Lipids = New Industrial Oils 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

“Second Wave” of Agricultural Biotechology • First wave considered to be agricultural traits e. g. herbicide and pest resistance, relatively simple single gene traits. • Concept of adding value to crops by using them as production systems for novel proteins

Overall Aims of Research • To use plants as bioreactors(enzyme factories) for the production of industrial enzymes including animal feed enzymes and cellulases for use in biomass conversion • To add value to an existing crop and recover other co-products by fractionation.

Currently most commodity enzymes are produced in capital intensive fermentation systems

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

Why Alfalfa ? • Abundant, widespread, hardy crop capable of 3 or more harvests a year • Low production costs, perennial • Legume capable of vigorous growth without irrigation and with less added fertilizer • Technology already developed to extract proteinrich juice from alfalfa on a large scale • Residue useable as feed, no waste management problems

Fields of Transgenic Alfalfa Will Replace Fermentation Systems

Production of the Animal Feed Enzyme Phytase in Transgenic Alfalfa 1. Swine and poultry need phosphorus (P) 2. P is present in seeds such as soybean and corn but it is bound in the form of phytic acid 3. Supplemental P is added to animal diets which is an added cost and leads to excess P in manure which causes environmental pollution If Phytase is added to feed the animals can use the P in the diet, less P in the manure

Alfalfa Transformation and Field Testing • We made transgenic alfalfa by inserting a gene from a fungus that makes phytase • We grew the alfalfa in fields, it was perfectly normal and the plants made high levels of the enzyme • We extracted juice from the plants and made leaf meal which we added to animal feed

Poultry Feeding Trials • Feeding trials using whole alfalfa juice and leaf meal preparations from phytaseexpressing alfalfa show that recombinant phytase from alfalfa performs as well as the microbial enzyme

Swine Feeding Trials Transgenic alfalfa juice and leaf meal preparations have both been shown to effective in swine feeding trials. Pigs actually like to eat alfalfa!

Phytase As a Feed Enzyme in Fish Farming • Expanding industry • Phosphorus supplementation required, especially if diet is grain based • Phytase effective in fish

Waste management is not a problem since the fibrous material remaining after juice expression is still a valuable ruminant animal feed or can be used as a substrate for bioethanol or fermented to lactic acid

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. Scott P. Bizily et al. , 2000 Mercurium Ionic mercuruim, easily eliminated BIOMAGNIFICATION Serious environmental threat as it enter chains

The problem? • Methylmercury is highly toxic!

Mercury Resistance • Organomercurial lyase (mer. B) • Mercuric Reductase (mer. A) Bizily et al, 2000

Hypothesis: • If plants are genetically engineered with the bacterial mercury resistance genes (mer. B and mer. A), 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, mer. A/mer. B-1, mer A, and wild-type plants A: 0 ppm CH 3 Hg. Cl B: 0. 2 ppm CH 3 Hg. Cl C: 1 ppm CH 3 Hg. Cl D: 2 ppm CH 3 Hg. Cl Bizily et al, 2000 (Figure 2 A-D)

Vaccine production in edible plants

HBs. Ag – major antigen of hep. B 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-Hbs. Ag antibodies titre in Mice fed with transgenic lupin callus 5 g in 1 dose 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 CT – inert component of cholera toxin (immunoboosting agent) Hugh S. Mason et al, 1998 Serum antibody responses of mice fed with Norwalk potato (4 g)

Human Trial of Norwalk vaccine 150 g of raw, peeled, diced potato (215 - 751 mkg of NVCP coat protein). 24 volonteers (20 exp + 4 controls) 19 out of 20 start to produce specific Ig. A antibody- secreting cells. 4 out of 20 start to produce specific Ig. G antibodies Side effects: nausea in 20% of all volonteers (raw potato) Titer of serum Ig. G anti-NVCP: 1 : 67 before immunization 1 : 757 after immunization among responders 1 : 62, 414 after real infection (fade after 2 years) Tacket et al. , The Journal of Infectious Diseases 2000; 182: 302 -305

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 ? SI SI può evitare facendo: -Integrare il gene nel DNA del cloroplasto (molte piante trasmettono i cloroplasti per via materna per cui il polline non è OGM); • Usare piante maschio sterili; • Non consentire coltivazioni non-OGM vicino ad 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 93 -96 %? 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.