Ch. 15

Question 1

How is mutation breeding performed?

Answer 1

Researchers expose a large number of seeds or totipotent tissues to the mutagen to generate various mutations in their DNA. The seeds are then planted and cultivated.

Question 2

Advantages/disadvantages of mutation breeding

Answer 2

• new colors and more petals have been expressed in flowers such as tulips, snapdragons, roses, chrysanthemums, and many others
• some of the varieties or food we eat today were developed using this method. For instance, peppermint plants that are resistant to fungus were generated this way
• the majority of seeds or tissues are killed by the treatment

Question 3

Transgenic plants -

Answer 3

A plant containing a gene (transgene) from a different plant or other organism

Question 4

What are totipotent cells?

Answer 4

• a cell that can develop into all the different types of cells of the body
• retain the ability to develop into fully mature plays from just one single cell

Question 5

Callus culture -

Answer 5

Dedifferentiating a mass of plant tissue in vitro and then growing the undifferentiated tissue in a petri dish

• the tissue can be an immature embryo, a piece of the apical meristem, or a root tip

Question 6

Suspension culture -

Answer 6

Dedifferentiating a mass of plant tissue and then growing the cells in a liquid medium

• cells must be dissociated from one another
• usually uses protoplasts, microspores, or macrospores

Question 7

Name 3 types of alterations in a plant regenerated from a single cell.

Answer 7

• temporary physiological changes
• epigenetic changes
• true genetic changes

Question 8

Temporary physiological changes -

Answer 8

Ex: When blueberry plants are regenerated via tissue culture, the plants are much shorter. These changes are not permanent, and after a few years of growing in the field, the regenerated blueberries are no different than any other blueberries.

Question 9

Epigenetic change -

Answer 9

• this is a modification that persists throughout the lifetime of the regenerated plant and may be passed on to a few subsequent generations, but the change does not permanently alter the cultivar
• often due to alterations in DNA methylation

Question 10

True genetic changes -

Answer 10

• affect the regenerated plant and all its progeny
• these changes may be due to point mutations, changes in ploidy level, chromosome rearrangements, activation of transposable elements, or changes to the chloroplast or mitochondrial genomes
• relatively common, but can be decreased by carefully regulating the available nutrients and hormones during tissue culture

Question 11

Describe the use of Ti plasmid in genetic engineering of plants.

Answer 11

• the most common cause for plant tumors is the Ti plasmid (tumor-inducing plasmid), which is carried by soil bacteria of the Agrobacterium group
• the most important aspect of the infection is that a specific segment of the Ti plasmid DNA is transferred from the bacteria to the plant
• because most DNA transfers occur only between closely related organisms, the ability of Agrobacterium to transfer DNA from one domain to another makes it an important tool for the genetic engineering of plants

Question 12

Study example in figure 15.5.

Agrobacterium transfers plasmid DNA into infected plants

Answer 12

Agrobacterium carrying a Ti plasmid is attracted by acetosyringone from a wounded plant stem. The Ti plasmid is cut by endonucleases to release single-stranded T-DNA, which is covered with protective proteins and transported into the plant cell through a conjugation-like mechanism. The T-DNA enters the plant nucleus, where is integrates into plant chromosomal DNA.

Question 13

Constitutive promoter -

Answer 13

• Functions in all tissues at all times

- turns gene on in all the plant cells throughout development

Question 14

Inducible promoter -

Answer 14

Acts as an on/off switch

Question 15

Study example of the two types of particle guns. Figure 15.10.

Answer 15

Particle gun A
- gene gun that operates via pressurized air

Particle gun B
- gene gun that operates via high-voltage discharge

Question 16

Particle bombardment -

Answer 16

A gun blasts microscopic metal particles carrying DNA through the tough plant cell walls.

Question 17

Reporter gene npt

Answer 17

• the simplest method to detect inserted DNA is incorporating the reporter gene npt, which encodes neomycin phosphotransferase
• this enzyme confers neomycin resistance by attaching a phosphate group to the molecule
• transformed cells are directly selected with the antibiotic neomycin, which kills any cells into plants using tissue culture techniques

Question 18

Reporter gene luc encoding luciferase

Answer 18

• this enzyme emits light when provided with its substrate, luciferin
• the luciferase protein is not stable for long in the plant, so the amount of active protein correlates with the level of gene expression at any given time
• therefore, luc can be used to determine the activity of specific promoters
• once cells successfully express the reporter gene are identified, they are regenerated into plants using tissue culture yevhbisheb

Question 19

Know the Cre/loxP system of bacteriophage P1

Answer 19

• bacteriophage p1, which naturally infects E. coli, has a simple system for genetic recombination, the Cre/lox system
• Cre stands for “causes recombination” and is a recombinase enzyme that recognizes a specific 34 base-pair DNA sequence, the loxP site
• including the Cre gene in the transgenic construct and expressing it when it is time to delete the unwanted DNA segment is used to remove selectable marker genes from the plant DNA

Question 20

How are the dsDNA breaks repaired?

Answer 20

• there are two main methods to generate the double-stranded DNA breaks for genome editing
• the first method relies on endonucleases or restriction enzymes that have very long recognition sequences. These are called homing endonucleases, zinc finger nucleosides (ZFNs) or TALE nucleases (TALENs)
• the second method to infuse DNA breaks into a genome at precise locations is done by the CRISP/Cas9 system. The nuclease is cas9, which has two DNA cleavage domains.

Question 21

What is the main advantage of these new techniques?

TALENs, CRISPR, ZFNs

Answer 21

Beginning to change how genome modifications are made in all organisms, but these techniques are particularly useful for plant engineering.

Question 22

Product development and regulatory approval

What agencies are involved?

Answer 22

• before being released to the public, transgenic plants must also adhere to government regulations
• to conduct field tests of the transgenic crop, scientists must notify the Animal and Plant Health Inspection Service of the USDA, which must approve the plan
• if the transgenic crop satisfies the FDAs guidelines, it is deemed to be “substantially equivalent” to its non-transgenic counterpart, but the FDA does not issue a formal approval
• the environmental protection agency (EPA) evaluates the transgenic crop for potential effects on the environment and on animals or insects that inhabit the farmers field

Question 23

How can biotechnology decrease herbicide use?

Answer 23

• herbicides cost the worlds farmers more than $14 billion each year
• despite this massive investment, around 10% of crops is lost due to weeds
• one problem is that many of the herbicides used do not discriminate between crops and weeds
• one solution is to make the crops resistant to the herbicide by genetic engineering

Question 24

How can biotechnology decrease pesticide use?

Answer 24

• insecticides are often more toxic to humans than are herbicides because insecticides target species closer to our own
• bacteria of the genus Bacillis produce spores that contain a crystalline, or Cry protein
• when insects eat Bacillus spores, the Cry protein breaks down and releases the Bt toxin (only harmful to insects)
• instead of spraying crops with Bt toxin, scientists have used transgenic technology to insert the Cry genes directly into plants
• almost 20% of the bases of the Bt toxin gene were altered to make the gene more plant-like in codon usage
• because such tweaks do not change the amino acids encoded, the toxin protein sequence was not affected by the procedure
• however, the rate at which plant cells made the protein greatly increased and gave another 10-fold increase in toxin production
• the toxin does not have to be sprayed, which reduces both the amount of work necessary and the potential for contaminating nearby fields
• planting Bt crops has resulted in a dramatic reduction in the amount of insecticides required

Question 25

Golden rice

Answer 25

• has vitamin A
• expressed the biosynthetic pathway for a vitamin A precursor called beta-carotene, and a humanitarian effort to provide the developing world with this transgenic crop is slowly underway

Question 26

How can biotechnology decrease toxins in plants?

Answer 26

• reduce the amount of toxins naturally found in plants or to prevent them from being infected with toxin-producing fungi
• Ex: fungus Fusarium causes head blight in barley, a crop that is fermented to produce beer. Fusarium releases a toxin called deoxynivalenol, more commonly known as “vomitoxin” because is presence in beer can induce vomiting. Developing barley varieties that are resistant to Fusarium infection is therefore desirable.

Question 27

How can biotechnology decrease toxins in the soil?

Answer 27

Using plants to clean up the environment by sequestering soil or water contamination is known as phytoremediation

Question 28

Phytostabilization -

Answer 28

Plants provide ground cover for a contaminated site by protecting against erosion due to wind and water. The use of transgenic could increase the root system or enhance tolerance to the contaminant.

Question 29

Phytodegradation -

Answer 29

The plant breaks down the pollutant

Question 30

Phytostimulation -

Answer 30

The plant stimulates microbes to degrade it

Question 31

Phytoextraction -

Answer 31

A plant assimilated the contaminant into its tissue, after which it is harvested and disposed of properly

Question 32

Phytovolatilization -

Answer 32

Pollutants are absorbed from the soil and released into the atmosphere, usually after being converted to a less toxic form

Question 33

Give an analogy between resistance to transgenic mutations and resistance to antibiotics.

Answer 33

Resistance to transgenic mutations

• nature has responded to transgenic crops in the form of Bt toxin-resistant insects and herbicide resistant weeds.
• the solution is to create more transgenic crop varieties and to implement farming practices, such as integrated pest management, aimed at reducing the spread of resistance

Resistance to antibiotics
- occurs when bacteria change in some way that reduces or eliminates the effectiveness of drugs, chemicals, or other agents designed to cure or prevent infections. The bacteria survive and continue to multiple causing more harm.