T1 - Introduction to plant tissue culture by Dennis J by kmb15358


									Steen - J7 Intro & History                                                                                     Page 1

T1 - Introduction to plant tissue culture by Dennis J. Gray and
Robert N. Trigiano
Chapter 1 is an orientation to the book and to define some terms using a minimalist approach - keeping it simple.
With its broad-topic chapters and extensive proven lab exercises covering many TC applications and plant types, the
book is designed for intro and advanced courses alike. The book assumes a good understanding of botany so it will
be helpful to have other botany texts near at hand.

How can we do this book justice since we are covering both plant and animal TC in a one semester introductory
course? Answer: we can't. But it is a good place to start.

The book's 5 sections are introduced:

1. History of Plant Tissue Culture: people, places, and dates of significant milestones that got us to where we are
now with special emphasis on how research was affected by world history. Ends with the "hot" topic of genetic
engineering and why it is seen as controversial.

2. Supporting Methodologies: what a TC lab should look like, how to prepare media, aseptic technique,
visualizing, photographing, documenting - all important in this section. The general how to section. Callus is
defined as an "unorganized tumor-like growth of cells".

3. Propagation Techniques: this core of TC explores a) culturing meristems (micropropagation), b) organogenesis
(making organs - shoots & roots - from cells not prone to make them, and c) nonzygotic embryogenesis (making
embryos from somatic cells or the like. This is the heart of the book where many good lab exercises

4. Crop Improvement Techniques: propagation techniques are put to good use in this section along with protoplast
manipulation, haploic culture, transformation, and cryopreservation - all for improving various characteristics of
important commercial crop species.

5. Special Topics: concepts that didn't conveniently fit in any of the previoius sections appear here including why
variation (genotypic and phenotypic) is enhanced by TC methods, fern TC, commercial plant production, clean
cultures, and plant pathogens.

Wow, there is lots to cover in this little book. We could do a whole course with just this book and not cover it all. We
have to keep our focus on this being an introductory course for both plant and animal systems. We are not trying to
learn it all but rather to learn basic procedures that we could apply to our own research.

Finally, two important listservers are introduced. Dr. Mark Galatowitsch at University of Minnesota runs the Plant-
TC listerv which lets plant tissue culture people stay in touch. Dr. Doug Powell at the University of Guelph runs the
Agnet which focuses on transgenics. I would encourage the class to subscribe to both and to plug in for at least a
few weeks. Listen to the chatter. Participate if you like. I will put the important words to define here at the end:

In Vitro
Iv Vivo:
Laminar Flow Hood:
Non-zygotic embryogenesis:

T2 History of plant tissue and cell culture by James D. Caponetti,
Dennis J. Gray, and Robert N. Trigiano
Steen - J7 Intro & History                                                                                     Page 2
Cut a plant up into little pieces, culture those pieces, then grow them back into a whole plant - that is what plant TC
is all about. When did that get started and what was the road to its success.

                   Gottlieb Haberlandt - late 1800 - early 1900 - first successful plant tissue and cell culture -
                   leaf mesophyll tissue and hair cells. Based his work on the work of many others before him.
                   Tissue & cells did not divide however because PGRs (not discovered yet) were absent in his
                   media. Got discouraged and did other things. 1902 proposed totipotency, in 1913 discovered a
                   component of phloem cells could promote cell division - first discovery of cytokinins.

                    Philip White (1934) cultured tomato root tips could be repeatedly subcultured with inorganic
salts and yeast extract (B vitamins). More developments were achieved as the need for vitamins and plant growth
regulators was learned. White added Thiamine

Then auxins were discovered - that discovery started with the Darwins, which went through a long history with
Went and Thimann before they were really classed as plant hormones in the 30s. TC went fast after this with
Gautheret and Nobecourt getting callus from Carrot root and then White from tobacco. Then all ground to a halt
during the war because of the shortage of supplies. Van Overbeek discovered the value of coconut milk (we now
know that has cytokinins in it) in embryo culture and Skoog got organ formation in tobacco tissue .

After the war (1950 +) and a slow resumption of TC activity, discoveries started to come faster and faster -
especially with more use of PGRs. Skoog and his various associates came to understand kinetin in its various forms
(adenine sulfate, benzyl adenine, benzyl amino purine etc) and value in promoting cell division. Then gibberellins
came on the scene and some in vitro work was done with them. Finally ethylene and abscisic acid rounded out the
PGR possibilities.

Mid 50s + Plant Developmental Biology got a huge boost from invitro studies. Discoveries were made much sooner
because of TC methods. New equipment, transportation, communication methods - all helped advance
understanding. HEPA filter were developed which made laminar flow hoods possible by the late 60s so sterile
culture could become routine.

Parallel studies between plant callus and mammalian (and human) cancer helped speed progress in the 50s and 60s.
Lots of cancer research money found its way to supporting plant studies.

Important developments: Somatic cells developing to embryos and their importance in genetic engineering (1958),
Murashige and Skoog developed a good culture medium (1962) now used extensively by the ornamental plant
industry. Haploid cell culture (mid 60s) helped plant breeders. Plant protoplasts were isolated in the 60s by
Cocking. Then restriction endonucleases were discovered in the 70 provided another important genetic engineering
tool and biotechnology was born - lots of genetic modification (GM) started happening. During the 70s and 80s
many biotechnology companies were started to work on varous GM crops. Commercial Acerage of GM crops went
from 0 in 1995 to millions of acres by 1998 and beyond, mostly in the US. Herbicide and insect resistance is the big
issue. GM has huge potential but it needs careful thought and controls.

F1 Historical Background by R. Ian Freshney
1.1 Historical Background - ANIMAL Tissue culture started with Harrison 1907 (studying frog [cold
blooded - no incubation needed] embryo nerve fibers). In Vitro methods allow for more control by the researcher,
you can see what you are working with better and control the environment much better. First 50 years of studies
were actual tissue culture (undisaggregated tissues). Dispersed cell culture came in later but is still called "Tissue
Culture". First disaggregated cells cultured in 1916 by Rouse & Jones with surgical passage. But dispersed cell
culture didn't get going good till the 50s when trypsin was used for subculture by Delbecco and then for cloning
single cells. In 1952 Gey got the first continuous cell line going called HeLa which was later cloned by Puck using
X-rayed feeder layers. Parker's use of antibiotics in 1961 made it possible to keep these cell lines going. In the late
50s and early 60s development of defined media made it possible to have serum-free media.
Table 1.1 shows all the key events in the development of animal tissue culture.
Steen - J7 Intro & History                                                                                      Page 3
Passage: subculture of cells
Cell Line: what cells are called after passage - first one was L929 mouse L-cells.
Cell Strain: what cells are called after selection and characterization.
Feeder Layer: cells that can't divide any more but that are cocultured with more sensitive cells
Defined Media: a media with every component known

Both Trigiano and Freshney elaborate on their loose use of the term Tissue Culture. They both point out what it
really means but how it is a generic term to refer to all types of culture from single cells through organ cultures and
in the plants even to whole plant propagation methods called micropropagation.

Organ Culture: 3D, undisaggregated with histological features at air liquid interface
Cell Culture: dispersed cells taken from any source by any disaggregation method
Histotypic Culture: cells have been reaggregated to recreate 3D structure
Organotypic: a histotypic culture that has several different types of cells to have the look and feel of tissue again.

Interesting that animal tissue culture got started with frogs tissues. Cold blooded means you don't
need an incubater. Amphibian cells grow easier than mammal because amphibians are known to
regenerate limbs when they are cut off. This gave the start to warm-blooded applications like
chicken eggs, then mouse embryos, and finally got to human tumors because those cells grew so
easily. Rodent tissue produced continuous cell lines much easier than human. But human tumors
can be cultured. Gey et al. (1952) cultured HeLa cells from Henrietta Lacks shown here >>>>>.
Read this very interesting article online http://www.jhu.edu/~jhumag/0400web/01.html
Hayflick learned that non-cancer cells have a finite lifespan Then lots of other human cells were
grown such as epidermal keratinocytes, bronchial epithelium and vascular endothelium etc. Insect
cells and amphibian cells, fish cells and other types is now coming along.

Figure 1.2 shows that tissue culture can help us understand 1) intercellular activity such as DNA transcription,
protein synthesis, ect, 2) intracellular flux or stuff moving around in the cell such as signal transduction and calcium
mobilization etc, 3) pharmacology such as how drugs work, what they attach to and interact with etc, 4) cell-cell
interactions such as how they cooperate, communicate, connect and affect each other, 5) toxicology such as what
kills them and in what doses and how they react to toxins, 6) tissue engineering such as propagation and
differentiation, how to make a matrix or scaffold to structurally organize cells, 7) genomics which is analyzing the
genes and studying how the genes move around, 8) immunology which involves knowing epitopes and understaning
how antibodies are made by hybridomas as well as figuing out cytokines and signaling process, and 9) cell products
learning how to make certain proteins (proteomics), understanding secretion, bioreactors, product harvesting etc.

Advances in tissue culture helped by: 1) need for large scale production of antiviral vaccines, 2) need to understand
cancer (neoplasia) research, 3) need to reduce animal testing. 4) need to fuse cells, 5) need to manipulate genes
(Human Genome Project), 6) need to make monoclonal antibodies, 7) need to study the cell, its interactions and
control mechanisms. Medicine uses TC for chromosomal analysis, study of toxic compounds, homografting and
reconstructive surgery, gene grafting, tissue engineering, in vitro fertilization etc.

It is truely incredible how much has been done in animal tissue culture. So many recent advances have involved
research involving various TC methodologies. This is a wonderful overview.

1.2 & (3) Advantages (and disadvantages) of Tissue Culture are spelled out in Table 1.2 and 1.3
and then described more fully in the text. Advantages are mainly the ability to control, see, and study. Disadvantages
are primarily cost, equipment, characterizing but also the fact that cells change. Variability is a problem along with
various types of contamination.

1.4 Major Differences In Vitro points out that, once cells are isolated, they start changing because
they no longer have the cell cell interactions as before but different selection pressures kick in. So one must be very
careful how TC data is interpreted.

1.5 Types of Tissue Culture include 1) Organ Culture, 2) Primary Explant Culture, and 3) Cell Culture.
Steen - J7 Intro & History                                                                                       Page 4

Organ culture maintains tissue characteristics (hystotype) as much as possible, cultures at the gas-liquid
interface on a raft, grid or gel. Usually several different types of cells and tissues function together here. Tissues
retain their differentiated properties. Little if any propagation happens because there is little if any cell division
happening. Each experiment requires a fresh explant making quantitation difficult

Primary Explant culture removes small pieces of tissue (explant) to a flask where the cells migrate out onto
the substrate (liquid/solid interfaace) to grow. Those cells that migrated may go on to become a cell culture over

Cell culture first disperses the cells through chemical or enzymatic means and then grows them on the surface or
in a suspension. This maximizes cell proliferation and there will be a significant growth fraction which means the
culture can be passaged thus starting a cell line. Getting a cell line going means that 1) we get lots more cells that
become 2) selected for their capacity to grow meaning that 3) the population of cells will become more and more
uniform as this selection proceeds so that 4) the line can now be characterized and called a cell strain. This line may
transform in which case the cells will become anchorage independent, start to pile up into a 3D mass or they can be
grown in suspension culture. Usually the cells do not transform but simple stay as a finite line, growing as
monolayers at the liquid/solid interface in a anchorage dependent lifestyle. These cell cultures are ideal for
quantitation, characterization, and replicate sampling but poor in terms of cell-cell interactions or cell-matrix

This chapter dives in so very very fast with very little introduction or definition of terms. That is an area that
Freshney could work on to make it much easier to start.

1. Compare & contrast the main driving factors behind the growth and development of PLANT vs ANIMAL tissue

2. The most common form of animal tissue culture currently is: a) organotypic tissue culture b) histotypic tissue
culture c) primary explant culture d) cell culture e) suspension culture

3. When plant tissue culture first got started there was limited success. Then a little later, huge success. What made
the difference?

4. Plant tissue culture got a big boost when cancer research began its boom because there was an idea that plant
callus was another form of cancer. Was this thinking accurate or not? Explain fully

5.Define tissue culture as it is narrowly defined and then compare that to the standard every day use of the term
tissue culture.

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