GEN 314

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GEN 314

• Gene Manipulation Lecture Seven
• Gene transfer to plants

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Plant genetic engineering

• Made possible through the use of vector systems
  based on the bacterium Agrobacterium tumefaciens
• Alternatively,
• transfection of plant protoplasts (plant cells
  from which the cell wall has been removed), or
• transfection by biolistic devices (e.g. gene
  gun)
• However, transgenic plant production is limited
  to a few species
• Agrobacterium-based gene transfer is efficient
  mostly with dicotyledonous (dicots) plants
• monocot-Agrobacterium interaction is not
  normally productive, i.e. under natural
  circumstances (review on other factors, see Cheng
  et al. (2004) In vitro Cell. Dev. Biol. Plant, 40
  (1) 31-45)
• the ability to regenerate plants from single
  isolated transfected cells limited to
  favourite experimental species including
  tobacco, tomato, petunia, etc

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Plant regeneration

• Tissue culture process of using small pieces of
  living tissue (explants) that are grown
  aseptically for indefinite periods on a nutrient
  medium
• For successful tissue culture results, start with
  explants rich with undetermined cells, e.g.
  cortex or meristematic cells because they are
  capable of rapid proliferation
• Commonly used explants include buds, root tips,
  nodal segments and germinating seeds
• placed on suitable culture (nutrient) media gt
  grown into undifferentiated mass known as a
  callus (capable of organogenesis and somatic
  embryogenesis)
• For plant cells to develop into calluses,
  nutrient
  medium must contain plant hormones, i.e. auxins
  (e.g. IAA),
  cytokinins (e.g. kinetin) and gibberellins
  
  (e.g. gibberellic acid, GA3)
• amounts used vary from different parts of the
  same
  plant and for the same explant from different
  genera

www.liv.ac.uk
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Plant culture types

• Originate from different starting points
• a single cell
• a (cellular) tissue or part of an organ
• an explant
• Culture types include
• embryo culture growing embryonic cells on
  media and allowing them to develop into plants
• organ culture meristematic tip
• cell culture calluses
• and obviously, callus culture

www.scq.ubc.ca
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Plant cell suspension cultures

• Obtained by transferring a callus to a liquid
  callus induction medium (contain all the required
  nutrients and elements to allow optimal growth,
  which acts to turn all cells into undiff. cells)
• Agitation using a shaker cell mass breaks down
  to give a suspension of isolated cells, small
  clusters of cells and larger aggregates
• Isolated single cells are also capable of
  division and proliferation may occur when placed
  in a suitable (conditioned) medium
• Preparing conditioned medium,
• culturing high densities of cells of the same
  or different species in fresh medium for a few
  days
• remove cells by filter sterilization
• resultant (conditioned) media contain
  essential amino acids (e.g. glutamine and serine)
  growth regulators (e.g. cytokinins)
• Single cells (previously grown on conditioned
  media) can be plated out on solid media, as with
  microorganisms, and will proliferate to give a
  callus

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Protoplasts

• Very useful for plant cell manipulations because
  under certain conditions they are capable of
  forming somatic hybrids (protoplast fusion)
• Can be produced from suspension cultures, callus
  tissue or intact tissues (e.g. leaves) by
  mechanical disruption or treatment with
  cellulolytic or pectinolytic enzymes
• pectinase break up cell aggregates into
  individual cells
• cellulase remove the cell wall proper
• Collect protoplast suspensions by centrifugation,
  wash in medium
  without enzymes and separate from
  intact cells and cell debris by
  flotation on sucrose
  medium
• protoplasts can synthesize new cell walls and
  divide
  when plated on nutrient medium (ca. 10 15 days)

ausubellab.mgh.harvard.edu
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Agrobacterium

• Agrobacterium is a causal agent of Crown gall
  disease in a wide variety of gymnosperms and
  dicots
• Crown gall disease a tumour that can be induced
  by inoculating wound sites with the Gram-negative
  soil bacterium A. tumefaciens
  (Smith and Townsend
  1907)
• crown gall tissue represents true oncogenic
  transformation (thus,
  callus tissue can be cultivated in vitro in the
  absence of bacterium
  and retains its tumorous properties)
• Properties include
• ability to form an overgrowth when grafted
  onto a healthy plant
• capacity for unlimited growth as a callus in
  tissue culture in media
  devoid of hormones
• synthesis of opines (amino acid derivatives
  not found in normal tissues e.g. octopine,
  nopaline, agropine or agrocinopine fam. members)
  crown gall tumour cells continue to synthesize
  opines in tissue culture and shoots or whole
  plants

kdcomm.net
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Opines

• Opine synthesis is a property that is conferred
  upon the plant cell when transformed with
  Agrobacterium tumefaciens
• the bacterium strain determines which type of
  opine is produced by the host plant
• The metabolism of opines is fundamental to the
  induction of crown gall disease in susceptible
  plants
• Bacteria induce the synthesis of opines (which it
  can catabolize) use as a source of energy,
  carbon and/or nitrogen
• Thus, A. tumefaciens has evolved a natural system
  for genetically engineering plant cells so as to
  subvert them for its own needs

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Tumour-inducing principle

• Bacteria is not required for transformation of
  the plant cells ? tumour-inducing principle must
  be involved
• Tumour-forming (i.e. virulent) strains of A.
  tumefaciens contains large-plasmids (140-235 kb)
  that may confer the virulence trait (Zaenen et
  al. 1974) confirmation,
• virulence is lost when bacteria are cured of the
  plasmid (e.g. grow the cells _at_37?C and not 28?C)
• cured strains lose the capacity to utilize
  octopine or nopaline
• virulence is acquired by avirulent strains when a
  virulent plasmid is reintroduced by bacterial
  conjugation (Bomhoff et al. 1976 Gordon et al.
  1979) nopaline strain plasmid introduced into a
  previously octopine strain avirulent strain gt
  avirulent strain can induce nopaline tumours and
  catabolize nopaline

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Ti-plasmids

• Plasmids are essential for virulence
  tumour-inducing (Ti) plasmids
• presence of plasmid in A. tumefaciens ?
  tumorigenic strain
• The genetic information specifying bacterial
  utilization of opines (and their synthesis by
  plants) is also plasmid-borne
• Plasmids in the octopine group closely related
  whereas in the nopaline group, highly diverse
• little DNA homology between the two groups
  except for four limited regions including the
  region containing genes responsible for crown
  gall formation
• To be continued.