Genetically Engineering Plants

1. Genetically Engineering Plants Riyanda N G (10198) Vina E A (10221) Arini N (10268) Suluh N (10302)

3. Overview of The Process There are five major steps involved in genetically engineering plants. These are DNA isolation, single gene cloning, gene designing, cell transformation, and backcross breeding. • DNA is extracted from an organism that has the desired trait. • The desired gene is located and copied. • The gene is inserted into a single plant cell using a transformation method. If the transgene successfully lands in the cells nucleus and is incorporated into one of the chromosomes, then the trait that it codes for will be expressed in the cell's offspring. • The cell multiplies and grows a new plant that contains the transgene in all of its cells. • Through backcross breeding the transgenic plant is crossed with a plant from a high yielding line. The resulting hybrids are the genetically modified plants that can enter the marketplace.

4. DNA Isolation • Inside all plants cells is a nucleus, the "brain" of the cell. The nucleus contains all of the information the cell needs. This information is stored on chromosomes, made up of tightly spiraled DNA.

5. DNA is composed of four different nucleotides: adenine (A), guanine (G), thymine (T), and cytosine (C). These nucleotides make up the genetic language of life. The order of the nucleotides encodes all of the cell's information. • A set of nucleotides that code for a particular protein is called a gene, and each chromosome contains thousands of genes. Since the proteins a cell produces are responsible for its specific traits, by changing the genes of an organism you can change its proteins, and therefore its traits.

6. Cloning Genes Gene cloning is used to locate and copy a specific gene from the entire DNA of an organism. For example, suppose the red gene in this bacterium needs to be extracted from the rest of the DNA in order to be added to a plant.

7. The DNA is removed from bacteria cells and isolated in a test tube. A restriction enzyme is added to the isolated DNA, and cuts the extracted bacterial DNA into gene-sized pieces.

8. In another test tube, extracted bacterial plasmids are cut using the same restriction enzyme. The cut plasmids are mixed with the gene-sized pieces of DNA.

9. The two combine to form recombinant plasmids. Some of the plasmids will recombine with themselves without picking up the bacterial DNA. These will be useless. Other plasmids will contain the gene of interest

10. Designing Genes • Each gene has three distinct regions: • Promoter - Signals how much protein to produce and when it should be made. • Coding Region - Encodes which protein to produce. In order, codons (sets of three nucleotides) are read by the cell, specifying the next amino acid that must be made and added to the chain. • Termination Sequence - Signals the end of a gene, preventing the cell from combining two or more coding regions.

12. Genetic engineers can alter or replace one or more of the three regions to design a gene so that it will be expressed in a specific way in a plant cell. Here is an example of different genetic modifications that can be achieved by manipulating the gene sequence.

14. Combining 35S with Bt. CRYIA and inserting this gene in corn will make all parts of the plant poisonous to the corn borer. Combining PEP Carboxylase with Bt. CRYIA will produce a plant that is poisons to the corn borer only if the pest eats the green parts of the plant. Corn stalks, silks, and late season plants that have slowed their photosynthesis will remain edible to the borer. • The same holds for the Round Up Resistance coding region. Combining it with 35S will produce a plant that is completely immune to Roundup.

16. Transformation • There are a variety of methods available for moving genes into recepient cells. The oldest method involves recombinant DNA. Newer methods involve microinjection, electro and chemical poration, and bioballistics.

17. Recombinant DNA • It is biological organisms like plasmids or viruses carry foreign genes into cells. • Plasmids are small, circular pieces of DNA found in bacteria cells that are capable of crossing membranes. Plasmids can be removed from bacteria. Circular plasmids are cleaved and new genes are inserted. The modified plasmid can then crosses cell borders and combine with the recepient cell's DNA along with the additional genes. • Viruses, infectious particles that contain genetic material, are also used to transfer genes to a cell's DNA. Desired genes can be added to a virus. The virus can then carry these genes into a recipient cell in the process of infecting that cell. The virus is disabled so that while it can carry a new gene into a cell, it cannot redirect the cell's genetic machines to replicate the virus and kill the cell.

18. Microinjection • It is simply injecting genetic material containing the new gene into the recipient cell. • Microinjection is not well understood. Somehow the injected genes find the host cell genes and incorporate themselves into them. In larger cells, like many plant and animal cells, microinjection can be performed with a fine-tipped glass needle.

19. Electro- and Chemical Poration • It is creating holes in the cell membrane to allow new genes to enter. • Pores are created either by soaking the cells in chemicals that open up holes in cell membranes, or by running weak electric currents through the cells, also to open pores in the membrane.

20. Bioballistics • It is Projectile method utilizing metal slivers to deliver genes to the interior of the cell. • Very small slivers of metal, much smaller than the cell diameter, are coated in genetic material. The coated slivers are propelled into the cell using a shot gun. A metal plate placed before the cells stops the shell cartridge, but allows the slivers to pass through and into the cells on the other side. Once inside the cell, the genetic material is taken to the nucleus and incorporated into the recipient's DNA

21. Plant Breeding • After obtaining a transgenic event, the genetic engineer hands the seeds over to the plant breeder. • Lines that survive transformation and tissue culture well are typically lower yielding than current elite lines. To make these transgenic lines marketable, plant breeders must use breeding techniques to transfer the transgene into a high yielding elite line.

23. First, the breeder obtains an inbred line by self-pollinating the transgenic line. Each plant cell in the transgenic inbred line now contains two copies of the gene. • The transgenic seeds produced are planted along with seeds from an elite inbred. • Due to a lack of hybrid vigor, both of the inbred plants that grow from these seeds will be smaller than current hybrids. • When the plants reach the proper stage, they are cross-pollinated.

25. The seed from this crop, the F1 seed, is harvested. All of the offspring have one copy of the transgene, as well as 50% elite and 50% non-elite genes. • The F1 seed is planted near another elite inbred seed. The plants grow. Due to hybrid vigor, the F1 plant is larger than the elite inbred, but it still contains many undesirable genes. • The F1 plant is mated back to the elite inbred. This process is known as backcrossing.

27. This seed, the backcross 1 (BC1) generation is harvested. The plants that grow from these seeds will have 75% elite genes, and half will contain the transgene. • Again, BC1 plants are grown along with elite inbreds. Those that express the transgene are cross-pollinated with the elite inbreds.

29. This process is continued until the plants contain at least 98% of the elite genesand the transgene. This takes approximately 5 to 6 generations.

31. Has genetic engineering of foods been done? A FlavrSavr tomato, engineered to be more appealing. Peter Beyer demonstrating golden rice, engineered to be more nutritious.