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Topic 11:Biotechnology and cropengineering.

Lecturer: MSc. Tong Thi Hang

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I. Introduction:

A. What is Biotechnology?

Biotechnology is any technique that uses organisms orsubstancesfrom thoseorganisms, to make or modify a product, to improve plants or animals, or to develop

microorganisms for specific uses.

The Office of Technology Assessment of the US Congress (1995).

In general, Biotechnology is a science which involves tools and techniques to

improve organisms (they can be animal, plant or even microorganism) for humanspurpose, and the product from those modifications can be rare or not even existence

in nature.

Nowadays, Biotechnologys application can be seen in every field of modern life,from chemistry, industry, pharmacy to environment, agriculture and life science

B. What is Crop Engineering?

For more than 10,000 years, farmers have

been experimenting with plants to find thebest seeds to grow plentiful crops.

Together with the development of human

civilization, the demand of food also

grows. Because of that, new technologiesand new crops were integrated, and that is

what we call crop engineering.

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With the participation of Biotechnology, crop engineering has changed dramatically.

Biotechnology allows scientists to move specific genes from one species to another to

produce changes. It also makes conventional plant breeding more efficient by allowing

scientists to select and transfer only genes for desired traits. Plants created using

biotechnology are generally referred to as genetically modified(GM) or transgenic plants.

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II. Principles:

To have new crops and products with precious characteristics, scientists use plants or

microorganisms as target and change them in purpose. We will have a general look in the

principle of biotechnology that they use to modify crop: Plant Biotech and

Microorganisms Biotech.

A/ PLANT BIOTECHNOLOGY

The field of plant biotechnology is concerned with developing ways to improve the

production of plants in order to supply the worlds needs for food, fiber and fuel. Inaddition, plants provide us with many pharmaceuticals and industrial compounds. As our

population grows, our needs also grow. To increase the quantity of crop production as

well as to produce specific characteristics in plants, biotechnologists are using selectivegene techniques. The two major methods of propagation are:

Plant tissue culture

Genetic engineering

1/ Plant tissue culture

Plant tissue culture is a practice used to propagate plants under sterile conditions,

often to produce clones of a plant. By this way, we can mass product plants with

selected characteristics in purpose.

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Plant tissue culture is a broad term that used to define 6 different types of in vitro

plant culture techniques are recognized and each type can result in a whole plant:

_Callus culture culture of differentiated tissue from an explantation that

dedifferentiates.

_Cell culture culture of cells or cell aggregates (small clumps of cells) in liquidmedium

_Protoplast culture culture of plant cells with their cell walls removed

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_Embryo culture culture of isolated embryos

_Seed culture culture of seeds to generate plants

_Organ culture culture of isolated plant organs such as anthers, roots, buds,

and shoots

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Micro- propagation

Desirable plants are cloned through tissue culture to produce genetically identical plants

in a process called in vitro clonal propagation (also called micro-propagation).

The plant that supplies the material, cells or tissues that is cultured is called the parent

plants.

Researchers and horticulturists have exploited plant regeneration to propagate large

numbers of plant originated from a single plant.

There are 4 stages in micro-propagationStage 1: initiation of explant culture the selection of explants , sterilization of tissue

surface to prevent contamination and transfer of explant to nutrient media

Stage 2: Shoot initiation (proliferation) multiplication of shoot tissue from explant onnutrient media.

Stage 3: Root initiation multiplication of root tissue from explant on nutrient media.

Stage 4: Acclimatization transfer of plants to sterile soil or other substrate undercontrolled conditions.

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2/ Genetic engineering

Genetic engineering of plants is rapidly becoming

a reality and plant gene transfer is now a fertile

field. Genetic engineering involves manipulationof the genetic material toward a desired end in a

directed and predetermined way.

This is alternately called recombinant DNA

technology or gene cloning. Strictly speaking,

to "engineer" means to design, construct and

manipulate to a set plan. Genetic engineering

uses the techniques of molecular cloning andtransformation to alter the structure and

characteristics of genes directly, through this we

can change the characteristics of plants and crops.

The basic technique is quite simple.

-Isolation of the gene of interest (or a piece of DNA) to be cloned.

-Insertion of the gene into another piece of DNA called a vector,

which will allow it to be taken up by bacteria and replicated

within them as the cells grow and divide.

-Transfer of the recombinant vectors into bacterial cells, either

by transformation or by infection using viruses.

-Selection of those cells which contain the desired recombinant

vectors.

-Growth of the bacteria, which can be continued indefinitely,

to give as much cloned DNA as needed.

-Expression of the gene to obtain the desired product.

In short, gene cloning or genetic engineering is essentially

the insertion of a specific piece of "foreign" DNA into a cell

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in such a way that the inserted DNA is replicated and handed

on to daughter cells during cell division. The basic mechanism

of genetic engineering is discussed below, followed

by some details regarding plant genetic engineering.

B/ MICROORGANISM BIOTECHNOLOGYIn general, this field of biotechnology also uses the similar technique with plant

biotechnology that is genetic engineering, but the target is microorganisms. Modifiedmicroorganisms can be used in different ways: transferring foreign gene into plant to

improve their characteristics, using as microbial pesticides, or cleaning the environment...

1/ Ice-nucleating bacteriaHeterogeneous ice nuclei are necessary, and the common epiphytic ice nucleation active

(INA) bacteriaPseudomonas syringae van Hall andErwinia herbicola (Lhnis).

Dye are sufficient to incite frost injury to sensitive plants at 5C. The ice nucleation

activity of the bacteria occurs at the same temperatures at which frost injury to sensitive

plants occurs in nature. Bacterial ice nucleation on leaves can be detected at about 2C,whereas the leaves themselves, i.e. without INA bacteria, contain nuclei active only at

much lower temperatures. The temperature at which injury to plants occurs is predictable

on the basis of the ice nucleation activity of leaf discs, which in turn depends on thenumber and ice nucleation activity of their resident bacteria. Bacterial isolates which are

able to incite injury to corn at 5C are always active as ice nuclei at 5C. INA bacteria

incited frost injury to all of the species of sensitive plants tested.

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2. Microbial pesticidesRecent development include the engineering of insect-killing virus

Using Bt for insect resistant plant.

3. Use of microorganisms to waste clean up

Biodegradation of lignin by fungi:Phanerochaete chrysosporium andPenicillium

chrysosporium are found to have capacity to degrade either the lignin pollutants or thesimilarly structural explosives as TNT, DNT

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III. ACHIEVEMENT:

Insect-resistant Crops (Bacillus thuringiensis,BT)

1/Introduction:

Bacillus thuringiensis, commonly known as Bt, is a bacterium that occurs

naturally in the soil. For years, bacteriologists have known that some strains of Bt

produce proteins that kill certain insects with alkaline digestive tracts. When these insectsingest the protein produced by Bt, the function of their digestive systems is disrupted,

producing slow growth and, ultimately, death.

Bt is very selective - different strains of the bacterium kill different insects and

only those insects. Strains of Bt are effective against European corn borers and cotton

bollworms (Lepidoptera), Colorado potato beetles (Coleoptera), and certain flies andmosquitos (Diptera). Bt is not harmful to humans, other mammals, birds, fish, or

beneficial insects.

Bt and Biotechnology: plants can be genetically engineered to produce their own Bt.

2/How does it work:

The Bt toxin binds to very specific receptors on the epithelial membrane of theinsect gut.

The toxin then forms channels in the membrane that leads to ion leakage and ultimately,

death of the insect. This mode of action explains the specificity of Bt (from the presenceof the necessary recep- tors) and also shows why the toxin needs to be eaten by the insect

to function).

http://en.wikipedia.org/wiki/File:Bacillus_thuringiensis.JPG

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The most common biological pesticides used include Cry proteins from Bacillus

thuringiensis (Bt), which is a soil bacterium. Bt has a vast collection of cry genes. Theencoded proteins vary widely with respect to their insect control efficacy and target insect

specificity. It produces crystalline inclusions containing these proteins during sporulation.

When ingested by insect larvae, these crystalline inclusions dissociate into monomers inthe alkaline insect gut. The monomers further undergo proteolytic cleavage which results

in an activated insecticidal protein. This protein binds to the larval gut lining and

damages it inducing an antifeeding behavior in the larvae and eventually death. The cryprotein binds to the larval gut lining at specific host-encoded receptors. One of the first

genes to be commercially exploited for trait development in crops is the Bt cry. This was

the advent of plant produced pesticides. A few genes like cry1Ac, cry1F, cry2Ab, havebeen effectively incorporated into plants for control of insect pests.

Not all genes are expressed in all tissues. If a promoter is used with theBtgene

inserted into a crop that is expressed in all tissue, then the trait is effective in all plant

parts.

A :. For most Bt transgenic events, theCaMV35S promoter is used, whichexpresses in all tissue, including the

pollen . When pollen from Bt corn driftsto other plants, it could result in thedeath of non-target insects.

B :An alternate promoter sequence to the CaMV35S is the phosphoenolpyruvate (PEP) carbpromoter from a plant gene encoding a photosynthetic enzyme. The result is, the Bt trawith this promoter will produce the protein only in cells that are actively making photosyproteins. Hence the root, tassel, or ear tissue in Btcorn are not expressing the Bt trait.

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In bio-technology :

Cutting out the gene of interest in the bacteria, itstotal DNA is isolated by RE.

Making an expression cassette : insert the genebegins is called the promoter and the end, theterminator

By putting the cassette into a plasmid, millions ofcopies of it can be made

Increasing in yield :

Golden rice

1.Introduction:

Golden rice is a variety of Oryzasativa riceproduced through genetic engineering to

biosynthesizebeta-carotene, a precursor of pro-vitamin A in the edible parts of rice. Thescientific details of the rice were first published in Science in 2000. Golden rice was

http://en.wikipedia.org/wiki/Oryza_sativahttp://en.wikipedia.org/wiki/Oryza_sativahttp://en.wikipedia.org/wiki/Ricehttp://en.wikipedia.org/wiki/Ricehttp://en.wikipedia.org/wiki/Genetic_engineeringhttp://en.wikipedia.org/wiki/Biosynthesishttp://en.wikipedia.org/wiki/Biosynthesishttp://en.wikipedia.org/wiki/Beta-carotenehttp://en.wikipedia.org/wiki/Retinolhttp://en.wikipedia.org/wiki/Science_(journal)http://en.wikipedia.org/wiki/Science_(journal)http://en.wikipedia.org/wiki/Ricehttp://en.wikipedia.org/wiki/Genetic_engineeringhttp://en.wikipedia.org/wiki/Biosynthesishttp://en.wikipedia.org/wiki/Beta-carotenehttp://en.wikipedia.org/wiki/Retinolhttp://en.wikipedia.org/wiki/Science_(journal)http://en.wikipedia.org/wiki/Oryza_sativa

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developed as a fortified food to be used in areas where there is a shortage of dietary

vitamin A In 2005 a new variety called Golden Rice 2 was announced which produces up

to 23 times more beta-carotene than the original variety of golden rice. Neither variety iscurrently available for human consumption. Although golden rice was developed as a

humanitarian tool, it has met with significant opposition from environmental and anti-

globalization activists.

Golden rice was created byIngo Potrykusof the Institute of Plant Sciences at the

Swiss Federal Institute of Technology, working with Peter Beyerof the University of

Freiburg. The project started in 1992 and at the time of publication in 2000, golden ricewas considered a significant breakthrough in biotechnology as the researchers had

engineered an entire biosynthetic pathway

2.The pathways produce beta carotent in rice:

Rice plants synthesise -carotene in vegetative tissues but not in the grain, all but

two steps of the biosynthetic pathway are present in the grain. By addition of only

two genes, phytoene synthase (psy) and phytoene desaturase (crtI), the pathway

is reconstituted and -carotene is consequently accumulated in the endosperm, ie

the edible part of the grain.

All plant tissues that accumulate high levels of carotenoid have a mechanism for

carotenoid sequestration including crystallisation, oil deposition, membrane

proliferationor protein-lipid sequestration. The non-carotenogenic starchy rice

endosperm is very low in lipid and apparently lacks any such means for

carotenoid deposition. Another restriction in Golden Rice could have been

precursor supply. Also, many people believed that the whole carotenoid

biosynthetic pathwaycomposed of many stepswas completely absent in the

endosperm.

http://en.wikipedia.org/wiki/Eatinghttp://en.wikipedia.org/wiki/Humanitarianismhttp://en.wikipedia.org/wiki/Humanitarianismhttp://en.wikipedia.org/wiki/Environmentalismhttp://en.wikipedia.org/wiki/Globalizationhttp://en.wikipedia.org/wiki/Ingo_Potrykushttp://en.wikipedia.org/wiki/Ingo_Potrykushttp://en.wikipedia.org/wiki/Ingo_Potrykushttp://en.wikipedia.org/wiki/ETH_Z%C3%BCrichhttp://en.wikipedia.org/wiki/Peter_Beyerhttp://en.wikipedia.org/wiki/Peter_Beyerhttp://en.wikipedia.org/wiki/Freiburg_Universityhttp://en.wikipedia.org/wiki/Freiburg_Universityhttp://en.wikipedia.org/wiki/Freiburg_Universityhttp://en.wikipedia.org/wiki/Eatinghttp://en.wikipedia.org/wiki/Humanitarianismhttp://en.wikipedia.org/wiki/Environmentalismhttp://en.wikipedia.org/wiki/Globalizationhttp://en.wikipedia.org/wiki/Ingo_Potrykushttp://en.wikipedia.org/wiki/ETH_Z%C3%BCrichhttp://en.wikipedia.org/wiki/Peter_Beyerhttp://en.wikipedia.org/wiki/Freiburg_Universityhttp://en.wikipedia.org/wiki/Freiburg_University

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The precursor molecule for carotenoid biosynthesis is geranylgeranyl diphosphate

(GGDP). Horizontal bars delimit the steps of the carotenoid biosynthetic pathway that

were overcome using the two transgenes phytoene synthase (PSY) and themultifunctional bacterial carotene desaturase (CRTI), rather than the two plant

desaturases PDS and ZDS.

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3.process of produce golden rice :

1.The plasmid is removed from thebacterium with aid of a RE.

2.Corn and the Erwiniauredovorasoil bacterium each have a gene

essential to the production of beta-carotein.The two gene are removedwith RE.

3.Two genes and the plasmid jointogether by ligase

enzyme.Recombinant

DNA is implanted into some

agrobacteriaWhich insert DNA into plants.

4.Rice embryo are put into a petridish with the agrobacteria.The

agrobacteria infect the embryos and

transfer the recombinant DNA to

rices DNA .

5.the rice is planted and grown in a

green house .Succesful plant

produce golden-coloured ricegrained that are rich in beta-carotene.