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
 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
 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
 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)
 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
 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
 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
 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
 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
 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
 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
 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
 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
 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
 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"
 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
 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
 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