RE: [DIYbio] Protoplasts

[Sebastian Cocioba]

I work with them often. Im currently working on a DIY protocol for easy
extraction. Its a bit difficult to keep them happy. Anything you had in
mind?

Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D From: Hege Tapio
Sent: 3/19/2014 9:33 AM
To: diy...@googlegroups.com
Subject: [DIYbio] Protoplasts
Does anyone work with protoplasts?
I am interested to get in contact with persons working with isolation
of protoplasts And regenerating plants from protoplasts.
Would be interested to learn about procedures And protocols.

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[Sebastian Cocioba]

I assume you are speaking of plant protoplasts (there are many kinds of
protoplasts). If you have the time, somewhere in the archives of the
DIY Bio group is a lengthy post I sent about plant protoplast isolation
in decent detail. The biggest issue everyone faces when working with
plant protoplasts is the constant osmotic pressure required to keep the
cells from bursting. Cell walls are built to be under pressure (turgor)
as a structural solution to growing against gravity. The end result is
a plant cell that has no compression-spring-like exoskeleton and is
prone to bursting. The go to method of maintaining this pressure
artificially is with Osmolality. If you add enough sugar to the medium,
the plant will have ample food and be under that correct pressure to
properly function until cell walls form. The issue there is that as the
cells eat the sucrose, the pressure drops. An alternative to sucrose is
the similarly osmotic sugar alcohol mannitol (or sorbitol, xylenol,
etc). It is metabolically inert meaning it won't get consumed but does
the same job. Issue there is now the cells have no food. The job of the
scientist is to monitor and maintain this osmotic pressure and feed the
cells at the same time. One normally measures the effectiveness
qualitatively using a haemocytometer or cover-slip pyramid under a
microscope and monitors the cells for roundness. Its not the most
scientific method of assaying viability (there are others like
fluorescene diacetate or Evans blue dye which are more quantitative)
but it gives a good idea of where you stand cell health wise. Cells
should be round, and neighboring media should be as free of other
cellular debris as possible. I've included a picture of some
protoplasts I isolated recently as an example.

Next is the isolation itself. Enzymes are expensive but buying from the
source saves you a fortune. Yakult the Japanese yogurt company sells
the enzymes at a fair but still very expensive $300/100g of each of the
two enzymes cellulase and macerozyme. You will need to empirically
determine proper concentrations of each. I generally start with 1%
Cellulase and 0.25-0.5% Macerozyme. These are crude fungal extracts
that have cellulases, hemicellulases, and pectinases which as a
cocktail digest most cell wall structures. Keep in mind that if you
want to regenerate cells everything must be maintained aseptic all the
way through. Not all plants respond well to protoplast culture and few
species have been established as successful regeneration systems from
protoplast starting material. I don't mean to be discourage you, just
letting you know from a ton of personal failure its not an easy task
and requires a lot of trial and error. The main focus of my research is
to develop a protocol for cheap protoplast biotech. Im making good,
albeit slow, progress and will be more than happy to share the info and
compare notes. I wish you the best of luck and am very excited to hear
that someone else on this planet wants to work with protoplasts :)