Protoplast Isolation and Culture

1
Protoplast Isolation and Culture

• Suggested reading Bhojwani 12-13, Vasil 4
• Applications
• direct DNA uptake
• for many spp., this was the only way to transform
  cells before the particle gun
• still useful for transient expression studies
• protoplast fusion to create somatic hybrids
• "wide crosses" where even embryo culture won't
  work

2
Protoplast Isolation and Culture

• Applications
• protoplast fusion to create somatic hybrids
• "wide crosses" where even embryo culture won't
  work
• Citopsis gilletiana (wild) x Citrus sinensis
• citrus sexually incompatible spp.
• wild relative has disease/nematode resistance
• somatic hybrid used as a rootstock

3
Protoplast Isolation and Culture

• Applications
• protoplast fusion to create somatic hybrids
• Solanum somatic hybrids
• S tuberosum dihaploids fused with wild diploid
  (S. chacoense)
• resulting somatic hybrid (4n) is backcrossed to
  S. tuberosum cultivars (also 4n)
• overcomes sterility due to ploidy differences
  between somatic and sexual hybrids

4
Protoplast Isolation and Culture

• Procedure for isolating protoplasts from tobacco
  leaves
• disinfest leaves and rinse in sterile water
• allow leaves to wilt slightly, remove lower
  epidermis by peeling with sterile forceps
• transfer leaf pieces to the surface of a solution
  of salts and 13 mannitol, let stand 25-30 min.
  (plasmolysis)
• pipet off plasmolyzing solution from beneath leaf
  pieces and replace with 20 ml enzyme solution
  (cellulase and macerase)

5
Protoplast Isolation and Culture

• Procedure for isolating protoplasts from tobacco
  leaves
• incubate 2-20 h (predetermine time by pretesting)
• place a solution of salts in 25 sucrose into a
  centrifuge tube (about 1/3 full)
• pipet enzyme/protoplast mix onto the top of the
  25 sucrose (solutions will form 2 separate
  layers)
• spin at 800g
• pipet off the band of protoplasts at the
  interface of enzyme and 25 sucrose into another
  tube

6
Protoplast Isolation and Culture

• Procedure for isolating protoplasts from tobacco
  leaves
• fill the tube about 2/3 full with 13 mannitol
• spin at 500g protoplasts should pellet at the
  bottom
• wash sev. times, then resuspend the last time in
  a small volume of liquid MS medium with 9
  mannitol
• carefully resuspend protoplasts and determine the
  concentration (protoplasts/ml) by counting in a
  counting chamber or hemocytometer

7
Protoplast Isolation and Culture

• Procedure for isolating protoplasts from tobacco
  leaves
• dilute to 1 x 105 protoplasts per ml
• plate protoplasts (various techniques)
• After plating
• cell wall formation
• wall starts to form immediately, takes 2-7 days
  to form a complete new wall
• loss of spherical shape is a visual indicator

8
Protoplast Isolation and Culture

• After plating
• cell wall formation
• only cells forming walls will divide
• cell division and callus formation
• plating efficiency is extremely variable
• PE no. of dividing colonies per field divided
  by no. of live protoplasts at plating
• after 2 wks, multicellular colonies form
• at 4-5 wks, macroscopic colonies can be
  transferred to solid medium

9
Protoplast Isolation and Culture

• After plating
• plant regeneration
• mini callus colonies are grown on a
  callus-induction medium
• callus is transferred to a regeneration medium,
  which will vary depending on whether regeneration
  is by organogenesis or somatic embryogenesis
• Media and plating techniques

10
Protoplast Isolation and Culture

• Media and plating techniques
• liquid medium
• sitting or hanging drops work well for small
  populations
• semi-solid medium (aka immobilization)
• mix with 2x agarose (at 40 C with 2x protoplasts
  in liquid medium)
• low-melting point agarose melts at 30-35 C, is
  better, less stressful on protoplasts

11
Protoplast Isolation and Culture

• Media and plating techniques
• semi-solid medium (aka immobilization)
• pipet out into a petri dish before agarose
  solidifies
• as agarose solidifies, protoplasts are imbedded
  at low density, allowing essentially
  "single-cell" selection
• entrapment in alginate beads
• protoplasts in Na-alginate are dropped into Ca
  solution, Ca-alginate gel forms around protoplast

12
Protoplast Isolation and Culture

• Media and plating techniques
• entrapment in alginate beads
• when cell walls are formed, gel can be dissolved
  using a citrates solution
• the advantage is less heat stress on the
  protoplasts
• nurse cultures

13
Protoplast Isolation and Culture

• Media and plating techniques
• nurse cultures
• nurse cells are irradiated and embedded in a
  feeder layer protoplasts placed on top
• alternatively, live nurse cells placed on medium,
  nylon membrane on top of nurse cells, protoplasts
  on the membrane
• conditioned medium

14
Protoplast Isolation and Culture

• Media and plating techniques
• conditioned medium
• fast-growing cells removed, the remaining
  "conditioned medium" is used for growing
  protoplasts
• Protoplast fusion and somatic hybrids
• the fusion process

15
Protoplast Isolation and Culture

• Protoplast fusion and somatic hybrids
• the fusion process
• electrofusion protoplasts are aligned in a
  special chamber, electric current is applied,
  opening channels in cell membrane
• PEG fusion protoplasts are coated with PEG,
  then incubated together where cell membranes
  fuse, channels begin to form
• after fusion, "fusion products" begin to "round
  up"

16
Protoplast Isolation and Culture

• Protoplast fusion and somatic hybrids
• the fusion process
• eventually, cell membrane between is dissolved
  and nuclei fuse into 1 nucleus
• in this type of fusion, cytoplasm is mixed
• types of fusion products
• parental types unfused protoplasts that develop
• homokaryons fusion product of 2 (or more)
  "like" protoplasts
• heterokaryons fusion of "unlike" protoplasts

17
Protoplast Isolation and Culture

• Protoplast fusion and somatic hybrids
• heterokaryons are the nascent somatic hybrids
• selection of heterokaryons strategies
• cell sorting (Cell Facility should be able to do
  this)
• parental protoplasts are differentially labelled
  with fluorescent dyes, one green, one red
• heterokaryons are stained yellow and can be
  sorted based on that trait
• selection after plant regeneration

18
Protoplast Isolation and Culture

• Protoplast fusion and somatic hybrids
• selection of heterokaryons strategies
• selection after plant regeneration
• e.g., fusion of Solanum tuberosum and S.
  chacoense
• somatic hybrids selected as calli at 6 wks they
  are more vigorous (initial selection)
• selection based on regeneration S. chacoense
  doesn't regenerate, the somatic hybrid contains
  an anthocyanin pigment