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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

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Applications


"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

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Applications


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 differencesbetween somatic and sexual hybrids

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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 leafpieces and replace with 20 ml enzyme solution

(cellulase and macerase)

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leaves

incubate 2-20 h (predetermine time by pretesting)

place a solution of salts in 25% sucrose into acentrifuge 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

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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 acounting chamber or hemocytometer

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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

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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

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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

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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, isbetter, less stressful on protoplasts

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semi-solid medium (aka immobilization)

pipet out into a petri dish before agarose solidifies

as agarose solidifies, protoplasts are imbedded atlow density, allowing essentially "single-cell"

selection

entrapment in alginate beads

protoplasts in Na-alginate are dropped into Casolution, Ca-alginate gel forms around protoplast

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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

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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

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conditioned medium

fast-growing cells removed, the remaining

"conditioned medium" is used for growingprotoplasts

Protoplast fusion and somatic hybrids

the fusion process

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the fusion process

electrofusion protoplasts are aligned in a special

chamber, electric current is applied, openingchannels 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"

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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

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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

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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

Documents

lec10.423.07[1]