Topic 11:

engineering.

Biotechnology and crop

Lecturer: MSc. Tong Thi Hang

I. Introduction:
A. What is Biotechnology?
Biotechnology is any technique that uses organisms or substances from those organisms, 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 humans purpose, 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?

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.

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. In addition, 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 selective gene 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.

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 liquid medium

_ Protoplast culture culture of plant cells with their cell walls removed

_ 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

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-propagation Stage 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 on nutrient 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 under controlled conditions.

2/ Genetic engineering Genetic engineering of plants is rapidly becoming a reality and plant gene transfer is now a fertile field. Genetic engineering involves manipulation of 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 and transformation 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

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 BIOTECHNOLOGY
In general, this field of biotechnology also uses the similar technique with plant biotechnology that is genetic engineering, but the target is microorganisms. Modified microorganisms 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 bacteria Heterogeneous ice nuclei are necessary, and the common epiphytic ice nucleation active (INA) bacteria Pseudomonas syringae van Hall and Erwinia 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 the number 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.

2. Microbial pesticides Recent 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 and Penicillium chrysosporium are found to have capacity to degrade either the lignin pollutants or the similarly structural explosives as TNT, DNT

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 insects ingest 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 and mosquitos (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 specic receptors on the epithelial membrane of the insect 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 specicity of Bt (from the presence of the necessary recep- tors) and also shows why the toxin needs to be eaten by the insect to function).

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. The encoded 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 in the 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 cry protein 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, have been effectively incorporated into plants for control of insect pests. Not all genes are expressed in all tissues. If a promoter is used with the Bt gene 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, the CaMV35S promoter is used, which expresses in all tissue, including the pollen . When pollen from Bt corn drifts to other plants, it could result in the death of non-target insects.

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

In bio-technology :

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

Making an expression cassette : insert the gene begins is called the promoter and the end, the terminator

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

Increasing in yield : Golden rice 1.Introduction:

Golden rice is a variety of Oryzasativa rice produced through genetic engineering to biosynthesize beta-carotene, a precursor of pro-vitamin A in the edible parts of rice. The scientific details of the rice were first published in Science in 2000. Golden rice was

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 is currently available for human consumption. Although golden rice was developed as a humanitarian tool, it has met with significant opposition from environmental and antiglobalization activists. Golden rice was created by Ingo Potrykus of the Institute of Plant Sciences at the Swiss Federal Institute of Technology, working with Peter Beyer of the University of Freiburg. The project started in 1992 and at the time of publication in 2000, golden rice was 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 (crt I), 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 proliferation or 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.

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 the multifunctional bacterial carotene desaturase (CRTI), rather than the two plant desaturases PDS and ZDS.

3.process of produce golden rice : 1.The plasmid is removed from the bacterium with aid of a RE.

2.Corn and the Erwiniauredovora soil bacterium each have a gene essential to the production of betacarotein.The two gene are removed with RE.

3.Two genes and the plasmid join together by ligase enzyme.Recombinant DNA is implanted into some agrobacteria Which insert DNA into plants.

4.Rice embryo are put into a petri dish 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 rice grained that are rich in beta-carotene.