PBIO 100 Lecture Notes

I. Plant Breeding (in Maryland - 100 genetic companies)

• A. Traditional method of improving plants - selective breeding
  • 1. Selection of desired characteristics: yield, palatability, resistance to disease and insects, aesthetic characters
    2. Hybridization - bringing together desired genes (by controlled mating) from two or more individuals; results in a combination of desired characters in the offspring - a hybrid
    • a. farmers would save seed from the best plants to grow next year

  B. Green Revolution - The example of successful crop improvement (dwarf, high yielding wheat varieties)

  • 1. Norman Borlaug, awarded the Nobel Prize in 1970 for his role in the Green Revolution
    2. Borlaug is remains concerned about the future noting in particular the international agribusiness control of genetic material

  C. Limitations

  • 1. Can only use genes from within one species or several closely related species or wild species, becoming a limitation due to loss of genetic diversity
    2. Takes many years to develop an improved variety

II. Recombinant DNA [Important Illustration] and the new Genetics: Genetic Engineering

• A. Introduction
  • 1. In the early 1970's a Moratorium on a certain type of research was called by those doing the work
    • a. legislation was introduced before the US congress which would require congressional approval for these experiments
      b. involves a newly developed technology of gene manipulation [Important Illustration]
      c. experiments were deferred for 18 months so that an assessment could be made regarding the potential danger of the research

    2. Technique: ability to construct new combinations of DNA molecules, which do not exist naturally= Recombinant DNA. Remember the Central Dogma of Molecular Biology?

III. Combines two different technologies: Restriction Enzymes and Bacterial Plasmids

• A. Restriction Enzymes [Important Illustration]
  • 1. The real basis for the recombinant DNA techniques [Important illustration]: Sequence specific DNases: Recognize short sequences of bases in DNA, and make a double stranded cut in the DNA molecule
    • a. function in bacteria- destroy foreign DNA which might enter the cell
      • 1) each bacterium has its own restriction enzymes

      b. each enzyme recognizes only one type of sequence

      • 1) sequence recognized is called a Palindrome
        2) reads the same on the two strands in the opposite direction
        

        3) example: G A A T T C
                    C T T A A G
        
            some enzymes cut straight across, and others make 
            a staggered cut.
        
                      G A A T T C
                      C T T A A G
        
                      G     AATTC
                      CTTAA     G
        
            Creates fragments of DNA, all with the same 
            overlapping ends.
        

    2. Value of restriction enzymes

    • a. each enzyme only recognizes the same palindrome regardless of the source of DNA
      b. so, fragments, from different sources, produced by the same enzyme, contain the same overlapping ends
      c. treat with ligase = permanently joined together = Recombinant DNA molecule

  B. Plasmids

  • 1. Extrachromosomal genetic elements in bacteria
    2. Closed circular DNA molecules
    3. Replicate independent of the chromosome = many copies/cell
    4. Contain genes controlling such things as fertility, and antibiotic resistance

  C. Molecular cloning

  • 1. The usefulness of the technology combines plasmid biology with restriction enzymes in the following way
    • a. join a DNA fragment from one source to a bacterial plasmid which has been cut once with the same restriction enzyme
      b. plasmid is now called a vector and carries a foreign piece of DNA
      c. recombinant plasmid is reintroduced back into a bacterial cell
      d. inside the bacterial cell, the plasmid will carry one or more antibiotic resistance factors, so that when plated on a medium containing the antibiotic, only the strain of interest will grow. Plasmid is replicated and the number increased greatly = MOLECULAR CLONING

IV. Actual gene transfer to plants: A primer on biotechnology

• A. Agrobacterium tumefaciens - a bacterium that causes crown gall disease in many plants; unique among pathogens because it causes the disease by transferring bacterial DNA from its plasmid into the plant's chromosome, causes tumors
  B. Tissue culture - used to regenerate an entire plant from a single cell; involves growing cells under sterile conditions with different nutrients and hormones; in some plants you can grow entire new plant from one cell: See this discussion
  C. Plant genetic engineering using Agrobacterium tumefaciens: method to have one plant species (tobacco - easy to grow, we know a lot about their breeding) express a gene from a different species
  • 1. Leaf disk method of plant transformation
    • a. punch out leaf disks
      b. incubate with Agrobacterium culture carrying foreign gene
      c. place disks on culture medium to induce regeneration and to select for transgenic plants
      d. root regenerated plants
      e. every cell in the transgenic plant has the foreign gene
      f. these plants pass genes on to their offspring

V. Applications of technology

• A. Getting a gene from one species into another and having it expressed
  • 1. Example - bacteria that makes human insulin, insulin extracted from cows, but gene not human/ now human gene in bacteria to produce insulin
    2. Example - tobacco plant that glows in the dark (luciferin gene from fireflies), transfer of gene not just from one species to another but from one kingdom to another

  B. Examples of application of gene technology in agriculture

  • 1. Herbicide resistant plants
    • a. in some areas, crops genetically modified for herbicide tolerance could decrease the amount of herbicide used and allow for no-till agriculture, which can minimize erosion
      • 1) Roundup - a herbicide that is effective, non-toxic to animals, short-lived in environment
        2) Research groups developed crop plants resistant to Roundup, notably soybean, corn, tomato, potato, wheat, cotton, alfalfa, etc.

    2. Insect resistant plants

    • a. Bt toxin gene for resistance to leaf eating insect larva. e.g.

    3. Controlled ripening tomato

    • a. two varieties of tomato now on the market engineered for delayed ripening
      • 1) one tomato variety has an extra gene - a reverse copy of the gene responsible for an enzyme that breaks down cell walls; as a result, the tomato softens more slowly
        2) the other variety has a gene that controls the enzyme necessary for the production of ethylene, one factor that makes a tomato soft
        3) ongoing research in other crops such as peaches

    4. Enhanced resistance to viral diseases

    • a. Crops bioengineered for pest resistance could increase yield, eliminate the use of several insecticides now derived from fossil fuel, and reduce health risks and groundwater contamination from pesticides
      • 1) used Agrobacterium to insert gene from viral protein into plant cells
        2) when regenerated, the plant (tobacco) produced this protein and were more resistant to the virus

VI. Engineered crop plants approved for use:

• A. cotton resistant to herbicide
  B. insect-resistant potato
  C. insect-resistant corn
  D. herbicide-resistant soybean
  E. virus-resistant squash
  F. Other examples
  • 1. Oil Seed Crops
    2. The plastic potato
    3. A discussion bioremediation

  G. Summary from Australia [REQUIRED READING] and a recent summary from the United States [REQUIRED READING]

VII. Advantages of gene technology

• A. Foreign genes (from outside species) can be introduced
  B. Potentially faster than traditional plant breeding
  C. Specific genes can be transferred; much more control than in traditional plant breeding

VIII. Possible benefits of gene technology in plants

• A. More nutritious foods
  B. Modified foods

IX. Other current and possible benefits of gene technology

• A. Already benefits in human health (human insulin).
  B. Genetically engineered fish that grow much faster than wild (growth hormone gene).
  C. Recombinant bovine growth hormone already enables cows to use feed more efficiently and produce more milk.
  D. Gene Therapy: Use this technology of gene transfer to correct genetic defects; any human diseases known to be due to a single gene defect; use to correct single gene defect and cure disease
  • 1. Severe Combined Immune Deficiency. Bubble baby. due to a single gene defect- adenosine deaminase
    2. Lesch-Nyhan- severe mental retardation, self destructive behavior. One brain enzyme missing
    3. Cystic Fibrosis- single gene, inhale DNA with normal gene

  E. The future of genetic research: One review

X. Pros and cons of genetic engineering

• A. Cons
  • 1. Herbicide-resistance or insect-resistance genes could spread from the engineered crops to wild relatives and create weeds that are especially difficult to control
    2. Some scientists fear that the USDA does not require sufficient precautions to prevent the spread of genes from engineered plants to their wild relatives in field trials
    3. Some bioengineered products could wipe out the major exports of some developing nations
    • a. example: a genetically altered bacterium is under development that produces vanilla flavoring; this could eliminate markets for the vanilla beans, one of Madagascar's major agricultural products
      b. bovine growth hormone too expensive for small dairyman, so can't compete with big companies

    4. Biological control may solve the problem, cheaper and more effectively, as shown with the work on the cassava mealybug by Dr. Hans Herren, winner of the 1995 World Food Prize

  B. Pros

  • 1. Almost 100 million people are expected to be added to the world's population each year for the next 30 years
    • a. some believe that without biotechnology, we won't be able to increase the availability of affordable basic food.
      b. although biotechnology has potential risks, starvation is worse

Other Sites of Interest:
All about Arabidopsis: "Guinea pig of the plant world" by Bryan Ness
Biotechnology and Agriculture
A series of short reviews; excellent summaries!
Access Excellence: WWW based teaching resources sponsored by Genentech, Inc.
Brief review and excellent definitions
Bio Online: Links to biotech information
BioBlox: Biotechnology Resource Center
A primer on molecular genetics but pretty technical
BioEthics: Links to resources on bioethics (little botanical)
Code of Conduct for Plant Biotechnology
A discussion of plant breeding, still a bit technical
CIMMYT: Where Norman Borlaug started the Green Revolution
Reviews of books and articles on the Green Revolution
Perils Amidst the Promise: Ecological risks of transgenic crops in a global market by Jane Rissler & Margaret Mellon -- An excellent review worthy of serious review

Last revised: 5/13/99 - Bottino