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Chapter 6 Plant Biotechnology

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Title: Chapter 6 Plant Biotechnology


1
Chapter 6 Plant Biotechnology
  • BTEC3301

2
INTRODUCTION
  • Plant biotechnology is a process to produce a
    genetically modified plant by removing genetic
    information from an organism, manipulating it in
    the laboratory and then transferring it into a
    plant to change certain of its characteristics .
  • Plant Biotechnology is a rapidly expanding field
    within Biotechnology.

3
INTRODUCTION
  • It chiefly involves the introduction of foreign
    genes into economically important plant species,
    resulting in crop improvement and the production
    of novel products in plants.
  • Today, biotechnology is being used as a tool to
    give plants new traits that benefit agricultural
    production, the environment, and human nutrition
    and health.

4
INTRODUCTION
  • The goal of plant breeding is to combine
    desirable traits from different varieties of
    plants to produce plants of superior quality.
  • This approach to improving crop production has
    been very successful over the years.
  • For example, it would be beneficial to cross a
    tomato plant that bears sweeter fruit with one
    that exhibits increased disease resistance.

5
INTRODUCTION
  • The red juicy tomatoes on shelves are results of
    genetic engineering.
  • For hundreds of years plant breeders have cross
    fertilised related plant, selecting combinations
    from the offspring that provide the plant with
    new characteristics beneficial to men.
  • Today plant biotechnology encompasses two major
    areas, plant tissues culture and plant genetic
    engineering.

6
What are transgenic plants?
  • A transgenic crop plant contains a gene or genes
    which have been artificially inserted instead of
    the plant acquiring them through pollination.
  • The inserted gene sequence (known as the
    transgene) may come from another unrelated plant,
    or from a completely different species

7
..What are transgenic plants?
  • Example transgenic Bt corn, for example, which
    produces its own insecticide, contains a gene
    from a bacterium.
  • Plants containing transgenes are often called
    genetically modified or GM crops
  • Desirable genes may provide features such as
    higher yield or improved quality, pest or disease
    resistance, or tolerance to heat, cold and
    drought.

8
What are transgenic plants?
  • Combining the best genes in one plant is a long
    and difficult process, especially as traditional
    plant breeding has been limited to artificially
    crossing plants within the same species or with
    closely related species to bring different genes
    together.
  • Corn and soybean to resist pest in US are in the
    forefront of Biotechnological revolution.

9
Methods of Introducing Genes into Plant/ Plant
Biotechnology Techniques
A. Selective Breeding
  • This is usually done by crossing two members of
    the same species which has dominant alleles for
    particular genes such as long life and quick
    metabolism in one organism crossed with another
    organism possessing genes for fast growth and
    high yield.

10
.A. Selective Breeding
  • Since both these organisms have dominant genes
    for these desirable characteristics, when they
    are crossed they will produce at least some
    offspring that will show ALL of these desirable
    characteristics.
  • When such a cross occurs, the offspring is termed
    a hybrid

11
Plant Biotechnology Plant Biotechnology Techniques
B. Cloning (Growing plant from a single cell)
  • Cloning is the process of creating an identical
    copy of an original.
  • In plant cloning is done by tissue culture
    methods.
  • Refer for more info on tissue culture
  • http//www.jmu.edu/biology/biofac/facfro/cloning/c
    loning3.htm

12
Plant Biotechnology Plant Biotechnology Techniques
C. Nature's way
  • Agrobacterium tumefaciens is a common soil
    bacterium that causes crown gall disease by
    transferring some of its DNA to the plant host.
  • The transferred DNA (T-DNA) is stably integrated
    into the plant genome, where its expression leads
    to the synthesis of plant hormones and thus to
    the tumorous growth of the cells.
  • After discovering this process, scientists were
    able to "disarm" the bacterium, put new genes
    into it, and use the bacterium to harmlessly
    insert the desired genes into the plant genome

13
Plant Biotechnology Plant Biotechnology Techniques
D. Cellular target practice
  • Many of the techniques used in plant
    biotechnology can be grouped under the headings
    of Plant Tissue Culture and Plant Genetic
    Engineering.
  • One problem with working with plants is that they
    have a cell wall - how do you get a recombinant
    plasmid to cross that barrier?
  • The following are some of the techniques used
  • 1.Microinjection of single cells

14
Plant Biotechnology Plant Biotechnology
Techniques D. Cellular target practice
  • 2. Biolistic gene transfer
  • In the "biolistic" (a cross between biology and
    ballistics )or "gene gun" method, microscopic
    gold beads are coated with the gene of interest
    and shot into the plant cell with a pulse of
    helium.
  • Once inside the cell, the gene comes off the bead
    and integrates into the cell's genome.

15
Plant Biotechnology Plant Biotechnology
Techniques D. Cellular target practice
  • http//www.Bio-Rad.com/images/gene_gun_delivery.gi
    f

16
Plant Biotechnology Plant Biotechnology
Techniques D. Cellular target practice
  • Model from BioRad Biorad's Helios Gene Gun

17
Plant Biotechnology Plant Biotechnology
Techniques D. Cellular target practice
  • 3.Electroporation of cells grown without a cell
    wall (protoplasts)
  • Plant cells could be "electroporated" or mixed
    with a gene and "shocked" with a pulse of
    electricity, causing holes to form in the cell
    through which the DNA could flow.
  • The cell is subsequently able to repair the holes
    and the gene becomes a part of the plant genome.

18
Plant Biotechnology Plant Biotechnology
Techniques D. Cellular target practice
  • 4. Agrobacterium-mediated transfer
  • This process is called Agrobacterium - mediated
    transformation. (Refer article PN Achar 2000).
  • The process "disarm" the bacterium, put new genes
    into it, and use the bacterium to harmlessly
    insert the desired genes into the plant genome.

19
Plant Biotechnology Plant Biotechnology
Techniques D. Cellular target practice
  • 5. Protoplast Fusion (Plant tissue culture)
  • Plant tissue culture is the cultivation of plant
    cells or tissues on specifically formulated
    nutrient enriched media.
  • Under appropriate conditions, an entire plant can
    be regenerated from each single cell, permitting
    the rapid production of many identical plants.

20
  • Plant Biotechnology
  • Plant Biotechnology Techniques
  • Protoplast Fusion (Plant tissue culture)
  • Protoplast is a cell without cell wall
  • Protoplasts are capable of fusing forms somatic
    hybrids of even genetically incompatible plants
  • Hybrid protoplasts are then regenerated by tissue
    culture into whole hybrid plants .
  • Boccoflower is a hybrid between broccoli and
    cauliflower. (Refer pg fig.6.2 in text book)

21
  • Plant Biotechnology
  • Plant Biotechnology Techniques
  • Protoplast Fusion (Plant tissue culture)

22
Plant Biotechnology Plant Biotechnology
Techniques D. Cellular target practice
  • 6. Leaf Fragment technique/ Leaf Disk Method For
    Agrobacterium Mediated Transformation of Higher
    Plants
  • Culture leaf disc in genetically modified
    Agrobacterium.
  • Soil bacterium Agrobacterium tumefaciens causes
    crown gall disease in plants.
  • This disease is characterized by the formation of
    tumors on plants after infection of wound sites
    by the bacterium.

23
  • Plant Biotechnology
  • Plant Biotechnology Techniques
  • 6. Leaf Fragment technique
  • The crown gall cells gain two genetic properties
    not possessed by normal plant cells.
  • These are the ability to grow in culture in the
    absence of externally added hormones, and the
    production of unusual compounds called opines.
  • These opines are used by the bacterium as sources
    of carbon and nitrogen. The bacterium therefore
    creates a niche in the crown gall which is
    favorable for growth.

24
  • Plant Biotechnology
  • Plant Biotechnology Techniques
  • 6. Leaf Fragment technique
  • The molecular basis for this disease was
    identified in 1974 with the discovery of a 200 kb
    plasmid called the Ti plasmid (tumor inducing).
  • Removal of the plasmid leads to avirulence, and
    re introduction restores virulence.
  • This Ti plasmid can be constructed with foreign
    genes include antibiotic resistance markers,
    genes for useful agronomic traits such as
    herbicide tolerance, virus resistance, and insect
    resistance.

25
  • Plant Biotechnology
  • Plant Biotechnology Techniques
  • 6. Leaf Fragment technique
  • The significant finding was that a 13 kb piece of
    the Ti plasmid is transferred into the genome of
    the recipient cell.
  • This piece of transferred DNA is called the T
    DNA.
  • The transfer of this T DNA to plant cells is the
    key step in using Agrobacterium tumefaciens as an
    agent for directed transformation and genetic
    modification of higher plants.

26
  • Plant Biotechnology
  • Plant Biotechnology Techniques
  • 6. Leaf Fragment technique
  • A growing number of plant species have been
    successfully transformed with foreign genes using
    such artificially constructed plasmid vectors. .
  • The bacterial plasmid serves as an ideal vehicle
    for transferring DNA. And this is done by
    culturing leaf discs in medium containing
    genetically modified Agrobacterium.
  • Major limitation of this technique is that
    Agrobacterium cannot infect monocotyledons e.g.
    corn but can easily infect dicotyledons e.g.
    potatoes, tomatoes, soybeans etc.

27
  • Plant Biotechnology
  • Plant Biotechnology Techniques
  • 6. Leaf Fragment technique
  • Refer pg 138 fig 6.4
  • for regeneration of plants from leaf disc with
    the aid of Agrobacterium tumefaciens.
  • Note
  • Gene gun an alternative method for inserting gene
    instead of Agrobacterium!!!
  • Assignment for next week The gene Guns Pg
    140fig.6.5

28
Plant Genetic Engineering
Plant Biotechnology
  • Plant genetic engineering involves the
    manipulation of genes at cellular and molecular
    levels. Using techniques of plant genetic
    engineering, it is now possible to isolate genes
    of interest form one kind of organism
    incorporating them into another, thus resulting
    in the permanent change inn the genetic make-up
    of the recipient.

29
Chloroplast Engineering
Plant Biotechnology
  • DNA in a cell chloroplast can also accept new
    genes.
  • Plant can be transformed with new traits using
    chloroplast DNA
  • Pollen DNA is separate from chloroplast DNA
    hence, transformed chloroplast cannot be released
    along with pollen DNA

30
  • Generally ripe tomatoes are red and juicy
    usually perish quickly if not refrigerated and
    mushy.
  • The FlavrSavrTM tomato (introduced in 1994by
    Calgene Inc.) can stay ripe for weeks!!!! Without
    perishing quickly. Why?
  • The Flavr Savr tomato is designed to ripen on the
    vine, with minimal softening and transported to
    consumers, a definite plus for grocery stores.

Plant Biotechnology Chloroplast Engineering
31
  • Ripe tomatoes produces the enzyme
    polyglacturonase(PG).
  • The PG enzyme is responsible for the breakdown
    pectin, a building block in cell walls, which
    gives tomatoes their firmness.
  • To slow down the softening process, the Flavr
    Savr employs antisense technology to block PG
    enzyme production.
  • Antisense technology is a method of gene
    silencing.

Plant Biotechnology Chloroplast Engineering
32
  • Antisense technology is a method of gene
    silencing.
  • The first step in this process involved the
    isolation of the PG gene from the tomato.
  • The first step is to clone the antisense DNA with
    the PG gene and insert this DNA into the plasmid
    of an agrobacterium.
  • The bacterium is introduced to plant cells which
    transfers the gene of interest into plant cells.
  • The cells with the plasmid are grown by adding
    specific hormones.

Plant Biotechnology Chloroplast Engineering
33
  • The re-generated plants will express the
    antisense DNA and when the mRNA is made through
    the process of transcription the sense mRNA will
    bind to the anti-sense mRNA. This interferes
    with protein production (PG enzyme in tomato).
  • Antisense is being developed for potatoes to
    resist bruising.
  • Refer fig 6.7The Flavr SavrTM tomato.

Plant Biotechnology Chloroplast Engineering
34
DNA
  • Plant Biotechnology
  • Plant Biotechnology Techniques
  • Summary of Antisense mechanism


Summary of Antisense mechanism

How enzyme is made?
PRODUCED
35
  • Plant Biotechnology
  • Plant Biotechnology Techniques
  • Summary of Antisense mechanism

What Happens When A Cloned Antisense DNA Is Added
To The Original DNA?


36
  • Plant Biotechnology
  • Plant Biotechnology Techniques
  • Summary of Antisense mechanism

DNA


How enzyme is made?
37
  • Plant Biotechnology
  • Plant Biotechnology Techniques
  • Summary of Antisense mechanism



When A Cloned Antisense DNA Is Added To The
Original DNA
38
References
Plant Biotechnology
  • http//www.biores-irl.ie/biozone/plants.html
  • http//search.yahoo.com/search?pSelectivebreedin
    geiUTF-8frFP-tab-web-tfl0xwrt
  • http//www.colorado.edu/che/BioProcess20Engineeri
    ng/Lecture208/protoplastfusion.htm
  • http//www.inform.umd.edu/genetics/leafdisc.html
  • http//dragon.zoo.utoronto.ca/jlm-gmf/T0501D/meth
    ods.html for gene slicing
  • http//dragon.zoo.utoronto.ca/jlm-gmf/T0101A/Enzy
    me.html for Flavr Slavr

39
Next Chapters
  • Practical Applications of Genetic Engineering in
    Plants
  • Vaccine for plants
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