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 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 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

Applications

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)

– 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– 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

– 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– 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

• 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– 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

• 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

– 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

• 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• fast-growing cells removed, the remaining

"conditioned medium" is used for growing protoplasts

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“• 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

– 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

• 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