Plant Regeneration

1
Plant Regeneration

• Suggested reading Bhojwani, ch 5-6, Vasil ch. 2
• Importance of a regeneration pathway
• propagation
• transformation
• regeneration of somatic hybrids
• cell selection/somaclonal variation
• haploids from anther/microspore culture
• secondary products

2
Plant Regeneration (contin)

• Totipotency
• basis for regeneration
• the ability of a (plant) cell to regenerate the
  whole plant
• Pathways of regeneration
• organogenesis
• embryogenesis

3
Organogenesis

• Defn de novo organ formation
• for practical purposes, we're most interested in
  shoots, because shoots can be converted most
  easily to plants
• shoots can form from
• differentiated cells directly
• callus cells indirectly
• in both cases, dedifferentiation is followed at a
  later time by (re)differentiation

4
Organogenesis

• PGRs are prob. the most important factor
  affecting organogenesis
• cytokinins tend to stimulate formation of shoots
• auxins tend to stimulate formation of roots
• The central dogma of organogenesis
• a high cytokininauxin ratio promotes shoots and
  inhibits roots
• a high auxincytokinin ratio promotes roots
  and/or callus formation while inhibiting shoot
  formation

5
Organogenesis

• The reality endogenous levels of hormones play
  an important role in organogenesis
• Consequently, there are lots of exceptions
• many herbaceous shoots require no added auxin to
  root (adventitious rooting from stem base)
• African violet (Saintpaulia ionantha) initiates
  shoots readily with 1 mg/L IAA and 0.08 mg/L BA,
  opposite to what the dogma predicts

6
Organogenesis (contin)

• Agricultural usefulness of regeneration by shoot
  organogenesis
• propagation many begonia and African violet
  cultivars can be propagated by inducing shoots on
  leaf pieces
• transformation useful in Agrobacterium-mediated
  transformation, particularly dicots
• protoplasts useful for regeneration of somatic
  hybrids and cybrids

7
Somatic embryogenesis

• Somatic embryogenesis is a useful regeneration
  pathway for many monocots and dicots, but is
  especially useful for the grasses
• Types of embryogenesis
• zygotic embryogenesis the result of normal
  pollination and fertilization
• somatic embryogenesis embryos from (cultured)
  sporophytic cells , that is embryos arise
  indirectly

8
Somatic embryogenesis (contin)

• The composition of the culture medium controls
  the process
• auxin (usu. 2,4-D) added causes induction, the
  formation of embrygogenic clumps or
  proembryogenic masses (PEMs) (induction medium)
• auxin is deleted and the clumps become mature
  embryos (maturation medium)

9
Somatic embryogenesis (contin)

• Culturing methods
• solid medium
• liquid medium (often called suspension cultures)
• Two cell types are usu. present in embryogenic
  suspension cultures
• large, highly vacuolate, freely dispersed cells
• small, densely cytoplasmic, aggregated cell clumps

10
Somatic embryogenesis (contin)

• Stages of development
• early cell division doesn't follow a fixed
  pattern, unlike with zygotic embryogenesis
• later stages are very similar to zygotic embryos
  (dicot pattern)
• globular stage (multicellular)
• heart-shaped stage (bilateral symmetry)
  bipolarity
• torpedo-shaped stage consists of initial cells
  for the shoot/root meristem

11
Somatic embryogenesis (contin)

• Three criteria that define a somatic embryo
• bipolar structure (shoot and root pole)
• no vascular connection to surrounding cells
• single-cell origin
• Potential applications to crop improvement
• haploids for breeding, mutant screening
• germplasm storage and preservation
• "artificial seeds"

12
Somatic embryogenesis (contin)

• "Artificial seeds"
• clonal propagation theoretically much faster,
  easier to mechanize than micropropagation
• the alfalfa model
• callus initiation (using NAA)
• embryo induction (using 2,4-D)
• embryo formation (deleting 2,4-D), addition of
  ABA "normalizes" development
• embryo "conversion" treatments to get
  greenhouse plantlets to 1st trifoliate leaf

13
Somatic embryogenesis (contin)

• Results have not lived up to the promise
• somatic embryos are highly susceptible to
  desiccation
• "conversion" rates are low
• shelf life is short
• encapsulation techniques have yet to be perfected
• Potential uses
• breeders might maintain elite genotypes more
  cheaply
• double-cross hybrid seed could be produced more
  efficiently