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Inactivation of Allergens and Toxins. Transgenic Plants for Food Security in the Context of Development Rome, 15-19 May 2009. Piero Morandini Dept. of Biology Milan University (Italy). The dangers of nature and food Gene inactivation strategies Manipulating crops (toxins) - PowerPoint PPT Presentation
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Inactivation of Allergens and Toxins
Piero Morandini
Dept. of Biology
Milan University (Italy)
Transgenic Plants for Food Securityin the Context of Development
Rome, 15-19 May 2009
Outline
The dangers of nature and foodGene inactivation strategiesManipulating crops (toxins)Manipulating crops (allergens)Transgenic vs. classical approachesConsequences & conclusions Most of the material presented is the work
published by other groups
Nature: a ‘mine field’Toxic substances abound in nature, both in cultivated and wild plants
With time we learned how to avoid some (or at least limit their intake), inactivate others through various processes:
Proper storage
Cooking (e.g. heat inactivation)
Food processing (e.g.: maceration, fermentation)
Knowledge handed through culture and technology (to detect, avoid & inactivate) are crucial for survival
Technology and knowledge buffer us from the toxic effect of nature
precise map metal detector
Plant Toxic substance Effect DoseAssa foetida Prenylated coumarins lethal
Datura stramonium Iosciamine/scopolamine atropine lethal 2 ng/ml blood
Tobacco Nicotine lethal 20g leaf
Solanum sodomeum Solasonine, solanidine. toxic
Ricinus (castor bean) Ricin / Ricinoleic acid lethal 1 seed
Tomato tomatine
Potato solanine lethal 3-6 mg/Kg
Cassava Cyanogenic glucosides lethal?
Soybean Protease / amylase inhibitors
Almond Cyanogenic glucosides lethal 20 seeds
Brassicas Glucosinolates toxic
A field trip into the ‘mine field’
http://kanaya.naist.jp/knapsack_jsp/image.jsp?word=C00002265
Solasonine
Solasonine accumulates in Solanum sodomeum
LD50=30 mg/kg
Poisoning and Toxicology Handbookby Leikin & Paloucek 4th edition, Informa Health Care, 2007ISBN 1420044796, 9781420044799
Solanine abounds in green parts, sprouts and diseased potatos
http://commons.wikimedia.org/wiki/File:Potato_sprouts.jpg
March 27, 1925 340-341
http://depthofprocessing.blogspot.com/2009/05/are-potato-peels-nutritious.html
Plant Toxic substance Effect ContentCotton Gossypol (terpenoid) Cardio/ hepatotoxic
Lathyrus s. oxalyl-diaminopropionic acid (ODAP) Neurotoxin /paresis 0.3-3.2%
Cassava Cyanogenic glucoside Blocks respiration 15-50 mg/kg
Beans Lectin, amilase & protease inhibitors Reduce nutrient absorption
Almond Cyanogenic glucoside Lethal 29 mg/kg
Apricot Cyanogenic glucoside Lethal 6 mg/kg
Poppy morphine Lethal?
Bamboo shoots Cyanogenic glucoside 1 g/kg HCN
Maize, peanut Aflatoxins Cancer 1-5 mg/kg
Maize Fumonisins Cancer / spina bifida
A matter of dose (threshold) reduction below a threshold considered safe
The issue of toxic substances in plants is not new nor is gone
Many crops still produce low levels of toxins (capacity is there!)
Their content may increase by breeding or spontaneously:e.g. Potato, Celery, Zucchini (courgette)…
Cultivated plants have less toxins than wild relatives (e.g. potato)
What happened? Mutants selected by human & animal tests
Trial and error (or trial and death)
http://www.springerlink.com/content/37827612x62xp348/
Bottle gourd Celery
http://www.people.cornell.edu/pages/kjc34/distribution.html
http://nonsense123.files.wordpress.com/2008/06/bottle-gourd.jpg
Zucchini Blackjack
http://www.growfruitandveg.co.uk/grapevine/vegging-out/courgettes-doing-my-head_18095.html
Which approaches?
How to reduce toxic substances in plant? Target the gene(s)
Gene RNA Protein
Toxins are either proteins or are produced through proteins
Gene inactivation strategies
Regulator
a) inactivate a protein (enzyme)
Halkier (2006) Annu. Rev. Plant Biol. 57:303-33
Degradation product
b) increase toxin degradation
c) target a regulator of the synthesis
Tools of the tradeto inactivate (plant) genes
‘Classical’ mutation (base change, insertion, deletion…)
Insertional mutagenesis (transposons or T-DNA)
RNA mediated (antisense, RNAi, miRNA, hpRNA…) collectively known as post-transcriptional gene silencing (PTGS), often involving epigenetic changes
Different methods may end up exactly in the same result (inactivation of a gene) and the same change at the DNA level
Method Pros Cons
Spontaneous mutation no/little regulat. slow / restricted choice
Induced mutation / cross no/little regulat. slow / restricted choice
Mutagenic oligonucleot. specific, quick, little/no regulat.
restricted choice
Transposon may be specific may be reversible, single target, restricted
T-DNA specific /irreversible
single target
Antisense quite specific, multiple targets
silenced gene not destroyed
RNAi (hpRNA) very specific/ multiple targets
silenced gene not destroyed
miRNA specific/ multiple targets
silenced gene not destroyed
Each method has pros & cons
None suites all situations
Hard or impossible distiguish natural / non-natural
Protein
toxin
Transcription
‘good’ phenotype
Direct gene inactivation
The gene is mutated (becomes non functional)
RNA missing or aberrant
Protein missing or non functional
Usually irreversible
‘Classical’ mutation Mutations arise spontaneously in any organism (endogenous or environment)
Frequency can be enhanced by various treatments: UV, X and γ-rays, chemical mutagens, and mitogens (indirectly)
Crop plants accumulated many mutations
Just one base change out of 15,000
Konishi et al., (2006) Science 312:1392-1396
Insertional mutagenesisTransposons: genetic elements able to jump around in the genome
Retroviruses: virus making new copies (through RNA) able to integrate into the genome.
T-DNA: bacterial DNA inserted into the plant genome
A large bit of DNA ‘breaks’ the gene
meaning is lost or altered
Inactivate genes by:
STOP
Gene = meaningful sentence
ATGT-DNA
Antisense RNA
Protein
toxin
Transcription
Duplex formation
Block of translationProtein
toxin
‘good’ phenotype
Indirect gene inactivation
The gene is intact but its expression inhibited
RNA is either missing, destroyed or non functional
Another gene is responsible for the change
Only knowledge of the sequence required
Transcription
Gene silencing
petunia expressing a maize gene
The presence of multiple copies of the maize gene causes partial or complete silencing of an endogenous gene
RNA-mediated
Mansoor et al., (2006) Trends Plant Sci. 11:559-65.
Once you know the sequence of a gene, it becomes easy to inactivate
- only that gene
- only in certain tissue/organ
Gene to be silenced
Gossypol, a problem
Cotton produces 1.65 kg of seed for 1 kg of fiber
Due to gossypol, a cardio- and hepatotoxic terpenoid seed unfit for consumption by humans and monogastric animals)
Seed contains 21% oil and 23% high-quality protein
Sunilkumar et al. (2006) P.N.A.S. 103:18054–18059
Cottonseed may help feed the world
Used as feed for ruminant animals (whole seeds or meal after oil extraction)
44 million metric tons (Mt) of cottonseed (9.4 Mt of protein)
Could fulfill protein requirements of half a billion people each year (50 g/day rate)
Sunilkumar et al. (2006) PNAS 103:18054-9
Proposed biosynthetic pathway of gossypol Sunilkumar et al. (2006) PNAS 103:18054-9
Chlorophyll
Carotenoids
…
RNAi inactivating δ-cadinene synthase in the seed
Levels of gossypol (mg/mg seed) for each individual seed
a strong reduction of gossypol in seed
Sunilkumar et al. (2006) PNAS 103:18054-9
Transgenic seed exhibits a large reduction in Gossypol level.
Sunilkumar et al. (2006) PNAS 103:18054–18059
A monogenic trait: the reduced gossypol trait cosegregates with the transgene Much more predictable, stable, specific
Transgenic vs. conventional
A glandless mutant was obtained with conventional strategies. Varieties with this trait were a failure under field conditions (extraordinarily susceptible to a host of insect pests)
Terpenoids protect the plant from both insects and pathogens
The transgenic approach achieved a goal classical breeding was unable to obtain (specific reduction in seed)
Sunilkumar et al. (2006) PNAS 103:18054–18059
Targeted gene silencing can be used to modulate biosynthetic pathways in a specific tissue to obtain a desired phenotype.Impossible by traditional breeding
Texas A&M University and U.S. Department of Agriculture
Take home message (I)“[this] approach…not only improves food safety but also provides an additional and potentially extraordinary mean to meet the nutritional requirements of the growing world population without having to increase either crop yields or acreage planted” (Sunilkumar et al., 2006)
“Our hope is to get through regulatory approval process in the U.S. first. However, it takes $50-100 million to go through the process. At this point, we don't know where the money is going to come from, but we are exploring various possibilities. Getting U.S. approval will make it easier to then get permit in other countries. We will be especially interested in some African countries and some Asian countries. “
(personal communication by Keerti S. Rathore)
Gene technology could improve food safety, food security and reduce environmental impact. Regulation is a major obstacle
Lathyrus sativus
A hardy tropical/subtropical legume
http://www.treknature.com/gallery/Asia/India/photo152618.htm
Important source of nutrition but contains a neurotoxin: oxalyldiamino-propionic acid (ODAP)
Beans from this so-called “famine crop” (consumed by poor people in Asia and Africa) causes lathyrism.
a paralytic disease (spastic paraparesis) prevalent among adults in Central India who have consumed large quantities of L. sativus seeds for several months
Safe content for ODAP is < 0.2%. Content in varieties range: 0.30-3.3
Classical breeding approaches are in progress, but what about a transgenic approach targeting the toxin biosynthetic pathway only in the seed?
http://www.grainlegumes.com/fckeditor/aepfiles/File/Species/Lathyrus_sativus_pod_(L.delaRosa)_600.jpg
http://www.gudjons.com/Mittel/Lathyrus-tub.jpg
Proposed biosynthetic pathway for β-1 in Lathyrus sativus (products in brackets have not been detected)
Yan et al., (2006) Phytochemistry 67:107–121
Biosynthesis
Fonio & pearl millet cause goiter
Gressel (2008) Genetic Glass Ceilings
Mycotoxins Mycotoxins in grains are a major health problem (fumonisins and aflatoxins)
Cause cancer and neural tube defects
- direct: improve resistance to fungi (corn expressing plant defensin in field trials with encouraging results)
- Engineer mycotoxin degrading activities- indirect: reduce insect damage through Bt toxin (effective for fumonisins)
Improve resistance or increase degradation
Courtesy of T. Maggiore
Courtesy of K. Petroni
Some colored lines show lower levels of fumonisin compared to control yellow lines
Tonelli, Pilu, Petroni (University of Milan, IT)
Classical breeding
(Flavonoid and anthocyanins)
Transgenesis
(Bt maize)
Courtesy of K. Petroni
Other examples by classical breedingErucic acid in Brassica napus
Cyanogenic glucosides in trifolium
Glucosinolates in brassica
Many food security/safety problems in the developing world are waiting for a solution. Genetic manipulation is
more precise, more predictable, low cost solutions
(not necessarily alternative to breeding)
Consequences of toxin reduction
Reducing glucosinolates in Arabidopsis
Glucosinolates are sulphur rich compounds from brassicas
Some beneficial, other toxic (quantity!)Upon wounding are converted into toxic
productsRegulators identified (two branches)Mutants isolated
Aliphatic GSL
Indolic GSL
Short chain
Long chain
Beekwilder et al., (2008) PLoS 3:e2068.
Mutating Myb28 and Myb29
Beekwilder et al., (2008) PLoS 3:e2068.
Regulators
Reducing glucosinolate content...
...stimulates pest growth and damage!
Beekwilder et al., (2008) PLoS 3:e2068
A. thaliana making cyanogenic glucosides
A) Adult beetles fed extensively only on leaves containing no dhurrin. B) Larvae frequently initiated no mines on leaves containing dhurrin, although attempts were made to feed (indicated by circles)
Effect on flea beetle and larvae feeding
Tattersall et al., (2001) Science 293:1826-8
Reduction in toxin content is a trade-off process: increases susceptibility to pests
Life is full of trade-offs
Reduction in pesticide content (a corollary of crop domestication) is not without consequences!
General pesticides (e.g. cyanide) are worse for humans than specific ones
Eat the pesticides you prefer (natural does not imply safer)
Take home message (II)
Allergens
Widespread occurrence You may not know it until you experience it Nuisance / cost / deadly threat Minute amounts of allergens may cause a life-
threatening anaphylactic reaction May occur after ingestion, skin contact, injection
of an allergen or inhalation. 48 deaths caused by food over a 7-year period
between 1999 and 2006 in UK
Anaphylactic shock: when food kills www.youtube.com/watch?v=XC0nHFblLcE
Allergies caused by plantsEight foods account for 90% of all food-allergic reactions. milk, egg, fish, shellfish, peanut, tree nut, soy, wheat
Contrary to common perception, transgenic plants never caused allergic reactions to consumers. Many conventional crops do it regularly
If a gene used for transgenesis comes from a plant containing allergens, the transgene is checked for allergenicity
Pollen is the major cause of respiratory allergy. At least 40% of type 1 allergic patients are sensitized against grass pollen allergens
Reducing plant allergens
Apple Peanut Wheat (celiac disease) Soybean Ryegrass Birch
Transgenesis, rather than a cause of allergy, can be part of the solution
Soybean allergen: P34US/Europe: 5 - 8% of babies and 2% of adults allergic* to soybeansDominant soybean allergens is P34: > 65% of soy-sensitive patients react only to P34 proteinTransgenic soybean without P34 published in 2003 (Herman et al., 2003)
No difference in composition, development, structure, or ultrastructure when compared with control plants. No other significant changes in polypeptide pattern
Herman et al., (2003) Plant Physiol. 132:36-43
Alternative approach: identify soybeans with little/no allergen screen the entire USDA national soybean germplasm collection
Out of > 16 266 accessions soybean germplasm screened, 12 lines (2 in the cultivated soybean) have no P34 allergen
Why these two soybean plants lack the antigen?
“Regulatory difficulties and the lack of acceptance of GM soybeans by the baby food and formula industry makes using such an allergen-suppressed soybean difficult [read impossible] at the present time.”
Joseph et al. (2006) 46:1755-63
Where logic ends, biotech regulation begins
If a protein is > 50% identical to an allergenic protein, it is a “potentially allergen”.
Transgenic products have to be labelled
Phaseolin ► eaten by one billion people everyday► NOT recognized as an allergen
BUT► 54% similar to conglycinin (a minor soybean allergen) ► ‘potential allergen’ according to biosafety regulation
Transgenic Cassava expressing phasolin produced by C. Faquet (Danforth) * improved (350%!) protein content* freely available (no royalties) in developing countries
but phaseolin similarity to a know allergen is 54% (>50%) transgenic cassava would require labelling (obviously impossible & ridiculous) in Africa
The glory of Him who moveth everythingDoth penetrate the universe, and shineIn one part more and in another less
(Dante, Paradise, Canto I, v.1-3)
The story of him who believeth everythingDoes perpetuate diverse lies and causesone part of farmers or another to die
60 % similarity does not give the same effect
Two texts with 60% similarity
New almond or peach varieties may accumulate much more cyanogenic glucosides, new potato varieties may accumulate more or new glycoalkaloids.
In Italy and the EU they require no regulatory scrutiny (no compulsory tests) before release, cultivation or commercialization if they are produced by conventional breeding or mutagenesis.
Conclusions
Plant derived allergens and toxins are ubiquitous, abundant
Tools are available to reduce them (conventional or transgenic)
Strategies must be reasonable (accept some level of risk) [risks / benefits]
Overcautious regulation kills the technology and associated benefits
Time to say the truthA man came home at noon, said good night to everybody, and went to bed. His wife, much concerned, asked him if he were ill, and the good man answered: 'I am quite well. But this morning everybody told me that I was drunk. I am not drunk -- you know that I do not even take wine, that I am an abstainer -- but I love peace, and in order not to contradict them I am going to bed.'
Plant biotechnologists avoided discussions (complied to insane regulations) for the sake of peace.
Time has come to raise the voice and demand a change
Present regulation is unscientific, very costly, excessive, restricting the potential uses of the technology to few crops and is aborting the technology in and for developing countries.Propagation of unreasonable and unfounded fears about biotechnology mantains a high regulatory burden and keeps de facto the technology in the hand of few industrial groups which are interested only in few crops/problems
The P38 and the apple
Un desiderio vero, quando non fa i conti con la realtà, ma imbocca la strada dell’utopia irrazionale, diventa menzogna, e non può che condurre alla follia omicida e all’autodistruzione.
S. Allevato e P. Cerocchi (2009) “La P38 e la mela”, ed. ITACA, p.173
A true desire, when does not come to terms with reality, but follows the path of irrational utopia, turns into lie and can only end up in murderous madness and self destruction.
S. Allevato e P. Cerocchi (2009) “La P38 e la mela”, publ. ITACA, p.173
Acknowledgements
• Support or material from colleagues and friends (especially Parrot, Gressel Kershen, Salamini, Fico, Vitale, Ederle, Maggiore, Petroni, Rossi…)
• Confidential information or help with literature (Gilissen, Bisht, Rathore, Shah, Carputo, Parisi, Faquet …)
• The support of the CSBA (access to specific books) and Univ. of Milan
Bibliography silencing/mutations
• Carlini and Grossi-de-Sa´ (2002) Toxicon 40:1515–1539• R Koes et al. (1995) Targeted gene inactivation in petunia by PCR-
based selection of transposon insertion mutants. Proc Natl Acad Sci U S A. 92:8149-8153.
• Hamilton AJ, Baulcombe DC (1999) A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286: 950–952
• Batista• Napoli et al. (1990) Introduction of a Chimeric Chalcone Synthase
Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans. Plant Cell. 2:279-289
• van der Kroll et al. (1988) An anti-sense chalcone synthase gene in transgenic plants inhibits flower pigmentation Nature 333:866-869
References classical mutants
* Li et al. (2006) Rice domestication by reducing shattering. Science 311:1936-1939.
* Konishi et al. (2006) An SNP caused loss of seed shattering during rice domestication. Science 312:1392-1396.
* Simons et al. (2006) Molecular characterization of the major wheat domestication gene Q. Genetics 172:547-555.* Wright et al. (2005) The effects of artificial selection of the maize genome. Science 308, 1310–1314.
* Peng et al., (1999) 'Green revolution' genes encode mutant gibberellin response modulators. Nature 400:256-61.
* Hedden P. (2003) The genes of the Green Revolution. Trends Genet. 19:5-9.
Dangers breeding/natural Lenape: Name of potato variety Lenape withdrawn . Am. J. Potato Res. (1970) 47:103
http://www.springerlink.com/content/a6k677974316541m/fulltext.pdf?page=1 Herrington (1983) Intense Bitterness in Commercial Zucchini. Cucurbit Genetics
Cooperative Report 6:75-76 http://cuke.hort.ncsu.edu/cgc/cgc06/cgc6-38.html Sharma (2006) Bottle Gourd Poisoning. Journal of Medical Education & Research, 8:120-
121. ISSN 0972-1177. http://openmed.nic.in/1710/01/r.letter.pdf Browning S, Hodges L. Cucumber crop information: Bitterness in Zucchini Squash and
cucumber. http://cuke.hort.ncsu.edu/cucurbit/cuke/cukehndbk/cukebitterness.html Finkelstein et al., (1994) An outbreak of phytophotodermatitis due to celery. Int. J.
Dermatol.33:116-8. Fleming (1990) Dermatitis in grocery workers associated with high natural concentrations
of furanocoumarins in celery. Allergy Proc. 11:125-7. Berkley et al., (1986) Dermatitis in grocery workers associated with high natural
concentrations of furanocoumarins in celery. Ann Intern Med. 105:351-5. Ames et al., (1990) Dietary pesticides (99.99% all natural). Proc. Nad. Acad. Sci. USA
87:7777-7781. Ames B.N., Profet M., and Gold L. S. (1990) Nature's chemicals and synthetic chemicals:
comparative toxicology. PNAS 87:19 7782-7786.
• Campion et al., (2009) Isolation and characterisation of an lpa (low phytic acid) mutant in common bean (Phaseolus vulgaris L.). Theor Appl Genet. 2009 Feb 18.
• Shi et al. (2007) Embryo-specific silencing of a transporter reduces phytic acid content of maize and soybean seeds Nature Biotechnology 25, 930-937
• Doria et al. (2009) Phytic acid prevents oxidative stress in seeds: evidence from a maize (Zea mays L.) low phytic acid mutant. J Exp Bot. 60:967-78.
Phytate has a negative impact on animal nutrition and the environment. Shi et al. produced soy and maize with a reduced phytate content
Phytate
Hansen A. A. (1925) Two fatal cases of potato poisoning Science 61:340-341
Lathyrus & MilletYan et al., (2006) Lathyrus sativus (grass pea) and its neurotoxin ODAP. Phytochemistry 67:107–121 (review)“Swollen Necks from Fonio Millet and Pearl Millet”, chapter 18 of Genetic Glass Ceilings (J. Gressel) 2008
* Sunilkumar et al. (2006) Engineering cottonseed for use in human nutrition by tissue-specific reduction of toxic gossypol. PNAS 103:18054-9* Siritunga and Sayre (2003) Generation of cyanogen-free transgenic cassava Planta 217: 367–373.* Beekwilder et al. (2008) The impact of the absence of aliphatic glucosinolates on insect herbivory in Arabidopsis. PLoS ONE. 3:e2068.
Other examples:* Lewis et al., (2008) RNA interference (RNAi)-induced suppression of nicotine demethylase activity reduces levels of a key carcinogen in cured tobacco leaves. Plant Biotechnol J. 2008 Feb 14;
Gene inactivation
Allen et al., (2004) RNAi-mediated replacement of morphine with the nonnarcotic alkaloid reticuline in opium poppy. Nat Biotechnol. 22:1559-66.
Liu Q, Singh SP, Green AG. (2002) High-stearic and High-oleic cottonseed oils produced by hairpin RNA-mediated post-transcriptional gene silencing.
Plant Physiol. Aug;129:1732-43.
Stoutjesdijk PA et al., hpRNA-mediated targeting of the Arabidopsis FAD2 gene gives highly efficient and stable silencing. Plant Physiol. 129:1723-31.
Grubb CD, Abel S. (2006) Glucosinolate metabolism and its control. Trends Plant Sci. 11:89-100.
Stevenson-Paulik et al., (2005) Generation of phytate-free seeds in Arabidopsis through disruption of inositol polyphosphate kinases. Proc Natl Acad Sci U S A. 102:12612-7.
Tattersall DB et al., (2001) Resistance to an herbivore through engineered cyanogenic glucoside synthesis. Science 293:1826-8.
Allergens• E. N. Clare Mills (Editor), Peter R. Shewry (Editor) "Plant Food Allergens“ (2005)
Wiley-Blackwell. ISBN-10: 0632059826
• Gilissen et al., (2005) Silencing the major apple allergen Mal d 1 by using the RNA interference approachJ. Allergy Clin. Immunol. 115:364-369
• Gao et al. (2008) Assessment of allelic diversity in intron-containing Mal d 1 genes and their association to apple allergenicity. BMC Plant Biology 8:116
• Herman et al., (2003) Targeted gene silencing removes an immunodominant allergen from soybean seeds. Plant Physiol. 132:36-43
• Herman E. (2005) Soybean Allergenicity and Suppression of the Immunodominant Allergen. Crop Sci. 45:462–467
• Schenk M. (2008) Birch pollen allergy: molecular characterization and hypoallergenic products. Ph.D. thesis (Wageningen University). ISBN 978-90-8504-873-2
• Pumphrey RS, Gowland MH. Further fatal allergic reactions to food in the United Kingdom, 1999-2006. J Allergy Clin Immunol 2007;119:1018-9.
• Anaphylactic shock: http://www.youtube.com/watch?v=XC0nHFblLcE• Yaklich, R., R. Helm, and E. Herman. 1999. Analysis of the distribution of the major
soybean allergen in a core collection of Glycine max accessions. Crop Sci. 39:1444–1447.
• Joseph L.M., Hymowitz T., Schmidt M.A. and Herman E. M. (2006) Evaluation of Glycine Germplasm for Nulls of the Immunodominant Allergen P34/Gly m Bd 30k. Crop Sci 46:1755-1763
Ref. toxicity
Cassava poisoning http://www.aciar.gov.au/project/CS2/1990/007http://www.cfs.gov.hk/english/multimedia/multimedia_pub/multimedia_pub_fsf_19_01.html Teles FF. (2002) Chronic poisoning by hydrogen cyanide in cassava and its prevention in
Africa and Latin America. Food Nutr Bull. 23:407-12.Oluwole et al., (2000) Persistence of tropical ataxic neuropathy in a Nigerian community. J.
Neurol. Neurosurg. Psychiatry 69:96-101.
http://www.foodstandards.gov.au/_srcfiles/28_Cyanogenic_glycosides.pdf (glucosidi cianogenici)http://www.ripper.com.au/~enneking/pdf/pdf-Famine-in-Afghanistan-threat-of-a-new-lathyrism-epidemic.pdf
(Lathyrism)
http://encyclopedia.farlex.com/cassava (Cassava)
Useful sites• www.botgard.ucla.edu/html/botanytextbooks/economicbotany/index.html
• Poisoning
• http://www.biomedexperts.com/Abstract.bme/17560071/Acute_Datura_stramonium_poisoning_in_an_emergency_department
• http://www.uni-sz.bg/tsj/Vo4No3_1/Binev%202.pdf
When too much becomes little
Eamens et al., (2008) Plant Physiol.147:456–468
RNA silencing of GUS in rice callus using a sense, antisense, or IR vector. Waterhouse et al. (1998)
PARENTAL TRANSGENIC
Napoli et al. (1990) Plant Cell. 2:279-289.
Petunia flowers in which the expression of a gene is modulated by introduction of extra copies
RNAi- mediated pathways in plants (2005)
Gene constructs. (A) Gene constructs Pro[s], Pro[a/s], and Pro[s]-stop. Pro[s] contains the Pro ORF in a sense orientation and is capable of expressing Pro protein, Pro[a/s] contains the Pro ORF in an antisense orientation, and Pro[s]-stop is the same as Pro[s] except that a thymidine residue has been inserted, making a stop codon (at the fourth codon) and a frameshift. (B) Constructs designed to express both Pro[s] and Pro[a/s] mRNAs. The Pro[s] is controlled in all constructs by the cauliflower mosaic virus 35S promoter; the terminators of Pro[s] and the promoters controlling the Pro[a/s] gene are shown. OCS, octopine synthase; term., terminator. (C) Constructs used to express ΔGUS mRNA in rice. The ΔGUS ORF has a 231-base deletion to prevent production of active GUS protein. With the exception of Gus[i/r] (i/r, inverted repeat), which is promoterless, the constructs are controlled by the ubiquitin promoter. UbiΔGus[s] and UbiΔGus[a/s] contain the ΔGUS ORFs in a sense and an antisense orientation, respectively. The 3′ region of the transcription unit of UbiΔGus[i/r] and ΔGus[i/r] is complementary to the 5′ region of the ΔGUS transcript. The predicted hairpin structure of such an mRNA is shown at the bottom.
From Waterhouse et al., (1998) PNAS 95:13959-64
RNAi construct for δ-cadinene synthase
T-DNA region of the vector used to transform cotton
Sunilkumar et al. (2006) PNAS 103:18054-9
Gossypol content of seeds in individual T1 lines
Levels of gossypol in pooled samples of 30 mature T1 seeds from 26 transgenic lines
Sunilkumar et al. (2006) PNAS 103:18054–18059
Individual transgenic seeds showed up to a 99% reduction in compared with wild type
No systemic reduction of gossypol and other protective terpenoids
Spatial and temporal confinement of RNAi-mediated suppression of the gene
Sunilkumar et al. (2006) PNAS 103:18054-9
Gossypol and other protective terpenoids are not reduced in leaves, floral organs, and roots
Sunilkumar et al. (2006) PNAS 103:18054–18059total heliocides (H)
Gossypol (G)
hemigossypolone (HGQ)
Effect on larvae
Nearly all larvae (98%) presented to leaves containing about 4 mg of dhurrin/gfw died.
Transgenic A. thaliana plants released high levels of HCN, up to 2 μmol/gfw, upon tissue damage.
An endogenous β-glucosidase with dhurrin hydrolyzing activity is present in A. thaliana.
Consumption of leaf-disc material from the transgenic lines expressing the two cytochrome P450 genes (CYP79A1 and CYP71E1), or the UDPG-glucosyltransferase gene (sbHMNGT), or containing the two empty expression vectors was not significantly different from the consumption of leaf-disc material from wt plants.
Tattersall et al., (2001) Science 293:1826-8
http://webschoolsolutions.com/biotech/transgen.htm
T-DNA mediated plant transformation
Cyanide in Cassava
http://www.food-info.net/uk/products/rt/cassava.htm
4th most important source of calories in the tropics. Staple for about 800 million people worldwide. Average annual per capita consumption of cassava in 2003 was 300 kg in Dem. Rep. Congo. Processing (and cooking) to reduce cyanide levels, but results in loss of proteins, vitamins, and minerals. Cyanide-associated health disorders have been attributed to eating poorly processed cassava
How to reduce cyanide content?
Siritunga et al., (2004) Plant Mol Biol. 56:661-9
http://www.fao.org/Wairdocs/ILRI/x5458E/x5458e0b.htm
http://thekebun.wordpress.com/2008/10/01/feeding-your-goats-cassava/
A strong reduction in root but not in leaf would be desirable
Transgenic plants show a strong reduction in leaf and root cyanogenic glucoside (linamarin) content
Siritunga et al., (2004) Plant Mol Biol. 56:661-9.
Transformants with large reductions (94% -60%) in leaf linamarin content all had root linamarin contents that were less than 1% of wild-type.
Apple major allergen: Mal d1Apple allergy is dominated by protein Mal d 1 where birch pollen is endemic inhibit the expression of Mal d 1 in apple plants transgenically by RNAi.expression successfully reduced reduced in vivo allergenicity
Allergenicity depends on the presence and amount of some specific Mal d 1 isoforms.
Classical breeding allows creation new hypo-allergenic cultivars
Gilissen et al. (2005) J. Allergy Clin. Immunol. 115:364-369
Gao et al. (2008) BMC Plant Biology 8:116