Genetic Engineering Technology Midterm 2 Lecture 5 And 6

Front 1

Plants are highly advanced (intro slide to plant biotechnology)

Back 1

Plants are the major energy transducers on Earth
• Plants are the base of our food supply
• Agriculture is the number 1 industry in the United States (and World)
• Biotechnological developments and genetic engineering methods are revolutionizing agriculture
-Tremendously increases the rate of progress
-Estimates suggest that ~70% of foods in the United States contain at least some GE crop plant

Front 2

what is plant tissue cell culture

Back 2

The sterile, in vitro cultivation of plant parts (e.g., organs, embryos, seeds, & single cells) on solidified/liquid media

-Meristematic tissue (capable of cell division; growing cells) is used to grow flowering plants only if it is virus-free, which is important for plant propagation
• Each season, potato and strawberry producers start with virus-free plants from tissue culture

Front 3

plant tissue cell culture facts

Back 3

-Much less controversial than animal tissue culture!
-Differentiated cells can be cultured to generate whole plants from very little starting material; no need for “embryonic”stem cells
-Large scale cloning is very simple and very common because all you need is a piece of the plant!
-The ability to give rise to a whole new plant through dedifferentiation and redifferentiation is called totipotency, and is unique to plants

Front 4

plant tissue cell culture steps

Back 4

1. Remove an explant (a piece of tissue from the plant)
2. Dedifferentiation–place explant in a specific ratio of nutrients and growth hormones to generate callus tissue in an meristematic state (plant “stem cells”)
3. Redifferentiation–transfer callus tissue into another nutrient medium to induce differentiation
4.Transfer the plant to soil to complete plant growth

Front 5

The six types of in vitro culture that can create a whole plant are

Back 5

1. Callus culture–culture of differentiated tissue from a whole plant through de-and redifferentiation (Fig 6.2)
2. Cell culture–culture of cells or cell aggregates (small clumps of cells) in liquid medium
3. Protoplast culture–culture of plant cells with their cell walls removed
4. Embryo culture–culture of isolated embryos
5. Seed culture–culture of seeds to generate plants
6. Organ culture–culture of isolated plant organs like anthers, roots, buds and shoots

Front 6

Micropropagation

Back 6

o Tissue culture is an excellent alternative way of propagating plants asexually
o The horticulture industry uses tissue culture extensively to produce aesthetically pleasing plants
o Desirable plants cloned through a process called “in vitro clonal propagation”or micropropagation
o Amounts of nutrients such as vitamins, sucrose, and plant growth hormones can control culture growth
• Altering the amounts of auxin and cytokinin induced multiple shoots to form from a culture
• Large scale production from a single plant = $$$

Front 7

Somatic Embryos

Back 7

o Method of plant tissue culture that produces embryo-like structures (embryoidsembryoids) from plant tissues
o Embryoids form directly from tissue by using hormones to disrupt normal development and to initiate embryoid formation
o Embryoids can also be formed from callus tissue
o Countless plantlets can be formed by this method
o Preferred method for large-scale commercial production
o Can use bioreactors to produce millions of embryoids (e.g., rice, sweet potato, potato, tomato, etc.)

Front 8

Protoplast Fusion

Back 8

oProtoplasts are plant cells that have had their cell wall enzymatically digested away
• Can be transformed with foreign genes via micro- injection & electroporation
oTwo protoplasts from unrelated plant species can be chemically fused or fused by electroporation
oThe genetic material of both cells is mixed together and the hybrid cell is screened for desirable traits
oMost hybrid cells yield hybrid plants with new traits

Front 9

Somaclonal Variation

Back 9

o Defined as the genetic variability produced by plant tissue culture
o Variability can be exploited to improve characteristics of crop & ornamental plants like corn, wheat, barley, and potato (see p.154)
o Resistance to stress (e.g., salt, heavy metals, insects) can be generated through a simple selection process
o Variability is caused by changes in chromosome number due to rearrangements, gene amplification (more of the same gene = more protein), etc.
o As a result, daughter plants differ from parent plant
o Keeping desired traits stable is key & may be very useful

Front 10

Germplasm storage

Back 10

o The genetic material of a plant may contain important characteristics (e.g., increase hardiness in plants)
o Ancient germplasm is often tolerant to a variety of insect pests, bacterial and fungal infection, etc. due to thousands of years of genetic selection
o Ancient germplasm is used to introduce new traits, such as insect resistance, into modern plants
o Germplasm is being lost due to the loss of traditional farming practices, clearing of old fields, and the use of modern plants in place of older plants
o Gene banks have been established in many countries throughout the World to preserve resources

Front 11

Genetic engineering in plants

Back 11

o The genetic engineering of crop plants is one of the most rapidly expanding areas of biotechnology
o While there are thousands of field trials underway, six different types of traits have been introduced into plants

Front 12

Genetically engineered plant traits: The Big Six

Back 12

• 1.Herbicide resistance
• 2.Insect resistance
• 3.Virus resistance
• 4.Altered oil content
• 5.Delayed fruit ripening
• 6.Pollen control

Front 13

Herbicide Resistance

Back 13

o Herbicides are a huge industry with use quadrupling between 1966 and 1991
o Plants that resist chemicals that kill indiscriminately are a growing need
o Critics claim that GE plants will lead to more chemical use because plants are engineered to be biochemically resistant to a specific chemical

Front 14

Glyphosate resistance

Back 14

• 1.Roundup TM inhibits an enzyme (EPSPS) that makes aromatic amino acids
• 2. Bacterial gene that breaks down Roundup TM is introduced into plants to confer resistance

Front 15

Glufosinate resistance

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• 1. Glufosinate mimics the structure of the amino acid glutamine and blocks the enzyme glutamate synthase
• 2.Plants receive a gene from bacterium Streptomyces that produces a protein that inactivates the herbicide

Front 16

Bromoxynyl resistance

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• 1.Gene encoding enzyme bromoxynil nitrilase (BXN) is transferred from bacteria to plants
• 2.BXN inactivates the Bromoxynil before it kills plants

Front 17

Sulfonylurea

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• 1. Kills plants by blocking enzyme synthesis of V, L, & I
• 2. A mutant tobacco gene confers resistance to plants

Front 18

Insect resistance

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o The Bt toxin from Bacillus thuringiensisis used in commercial crop plants such as corn, cotton, and potato
o Plant protease inhibitors have been explored since the 1990’s
• Protease inhibitors are naturally produced by plants in response to wounding
• They inhibit insect digestive enzymes after the pest has ingested the plant causing starvation
• Tobacco, potato and peas have been engineered to resist insects such as weevils
• Results haven’t been as promising as Bt toxin; Why?
• Insects have evolved resistance to PI’s

Front 19

Virus resistance

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o Chemicals are use to control the insect vectors of viruses, but controlling the disease itself is difficult because it can spread so rapidly
o Plants may be eng’d with genes for resistance to viruses, bacteria and fungi
o GE virus resistant plants harbor a viral coat protein gene that is overexpressed
o The plant shuts it off to conserve resources
o Because it shuts down the viral coat protein gene, it actively prevents the formation of viral coat proteins—either from it’s transgene or from an infecting virus
o This is called coat--protein mediated viral resistance
o Coat protein genes are involved in resistance to diseases such as cucumber mosaic virus, tobacco rattle virus, and potato virus X
o Yellow squash, potatoes and papaya have been genetically engineered to be virus resistant

Front 20

Altered Oil Content

Back 20

o Done in plants by modifying an enzyme in the fatty acid synthesis pathway (oils are lipids, as are fatty acids)
o Varieties of canola and soybean plants have been GE’d to produce oils with better cooking and nutritional properties
o GE’d plants may also be able to produce oils that are used in detergents, soaps, cosmetics, lubricants and paints
o Proctor & Gamble Company introduced the no-calorie fat substitute olestra® in 1998; critics cautioned that it would deplete the body of fat soluble vitamins
• It had other issues...

Front 21

Delayed fruit ripening

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o Allows for crops like tomatoes to have a longer shelf life
o Tomatoes generally ripen and become soft during transport
o Tomatoes are usually picked before they are ripe and later sprayed with ethylene to induce ripening
• This method does not allow flavor to develop
o Tomatoes have been engineered to produce less ethylene so they can develop more taste before ripening
o GE tomatoes are not on the market simply because they are not cost effective yet (e.g. The FlavrSavr Tomato)

Front 22

Pollen controll

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o Hybrid crops result from crossing distantly related varieties of the same crop plant
o This method may generate plants with favorable traits
o For success, plant pollination must be controlled
• Why? To ensure that the correct parental plants are being used in the cross
• Usually done by removing the male flower parts by hand before pollen is released
• Sterilized plants have also been GE’d with a gene from the bacteria Bacillus amyloliquefaciens

Front 23

Genetically engineered food

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o More than 60% of processed foods in the US contain ingredients from GE’d plants
o 12 different GE’d plants have been approved in the US, with many variations of each plant (some approved, some not)
o While already on our store shelves, GE’d food is still very controversial
o Public fears that genetically engineering a plant may produce unexpected results (e.g. allergies, shock, etc.)
o GE crops spreading out into and damaging local ecosystems
o Antibiotic resistance markers may inactivate antibiotics
o GE’d food also raises concerns about food choice
• How will a vegetarian respond to an apple with an animal gene?
• How will Muslims or Orthodox Jews respond to a pig gene in a vegetable?