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FUTURE USES OF P L A N T CELL CULTURE TECHNIQUES

PETER D SHARGOOL
D e p a r t m e n t of Biochemistry
University of Saskatchewan
Saskatoon, C a n a d a S7N O W O
Introduction The large-scale culture of plants for food, drugs, and other uses, faces a number of serious
challenges both now, and in the future. Thus we see agricultural land becoming scarcer,
and plant species disappearing, as the last remaining forests are destroyed by slash-and-
burn techniques. In addition, man continues to modify the environment in various ways
likely to reduce the growing capacity of at least some of the land area in the forseeable
future. Many scientists have turned to plant tissue culture as a means towards solving
many of the problems existing with whole plant culture. This article reviews some aspects
of plant tissue and cell culture, with a particular emphasis on future biotechnologicai
applications.
Plant Tissue Culture The first step in starting a plant tissue culture is the removal of an organ or piece of tissue
from a parent plant. This is then sterilized to remove surface microorganisms, and placed
in or on a medium that will nourish its further growth. This medium may be entirely liquid,
or solidified with agar. In both cases suitable mineral salts, vitamins, and a carbon source
such as sucrose, will be present in this medium. Texts which describe the manipulations
necessary to start such cultures, and the composition of various well known media, have
been published in paperback editions. 1'2 In liquid medium, plant cells tend to grow as a
fine suspension consisting of very small groups of cells with perhaps a few to a dozen cells
in a group. On solid medium a mass of cells (called a callus) will be formed. In both cases
the cells will remain undifferentiated and liquid cultures can be started by adding a piece
of solid callus to liquid medium, and vice versa for callus cultures (Fig 1).

Agar i ~ ~ Liquid
medium ~:.~ medium
Sterile Explant Callus Suspens=on
cultures
tissue

Figure 1 Procedure for starting callus and cell suspension cultures (redrawn from reference 1)

Tissue culture techniques have great potential in the realm of biotechnology. For
example organized juvenile tissue of several species such as date palm and carnation have
been successfully recovered after months of storage in liquid nitrogen. 3 This type of
technique thus offers potential for the preservation of germ plasm of threatened plant
species. The long-term goal of many industries such as the forest industry is to induce
single cells or cell aggregates in a liquid medium to associate into embryos. In this process
(termed somatic embryogenesis), the structures develop a shoot and root simultaneously.
The feasibility of this approach has already been demonstrated for important species such
as Douglas Fir. 4 It will be readily appreciated that using this technique, special or 'super'
trees have the potential to yield many similar cloned offspring (Fig 2) and could thus form
the basis of an efficient forestry industry. 4
Probably the most exciting aspect of plant cell culture techniques is their use to develop
plant cells with new characteristics not evident in the parent plants from which cultures
were derived. It is this aspect, plus the fact that if desired, such new and novel cells can
frequently be caused to regenerate whole plants, that gives plant cell culture great future
potential for industry.
Development of Whole plants can be regenerated from single plant cells or pieces of callus by suitable
new types of plant cells: manipulation of plant hormone levels in the growth medium. ~,2 While this process cannot
Mutagens and always be carried out successfully with every type of plant cell culture, the number of
somacional variation instances of success increases daily. In the past, workers have sought to develop plant cells

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Figure 2 A possible scenario for the next century's forests. Note the recurrent breeding cycle from
first-generation flowers to second generation progeny test, the cloning, by embryogenesis in
liquid culture, from buds from genetically superior trees, mechanical sowing in extruded gel
in protected nursery beds, monoclonal-block plantations, intensively managed, containing
densely-packed trees using advanced fertilization and irrigation practices, and lower
intensity, mixed-genotype forests
with new and novel properties by the use of mutagens such as BUdR.1 In recent years,
however, it has been realized that when plant tissues are taken along the route: tissue --->
callus ---> liquid culture ---> plants, the plants finally produced may include a number
with new properties of importance to industry, for example higher yield, larger size, and
disease resistance. It appears as if the trip through the de-differentiated state of tissue
culture has liberated new potentials previously locked within the plant cells. This
phenomenon has been termed somaclonal variation.5 The number of variants produced by
this method can in fact be so great that workers involved in breeding disease-free sugar
cane have noted that no more variation could be introduced into sugar cane cells by the
use of a mutagen, than already appeared to exist as a result of somaclonal variation. 5
Somaclonal variation has been used not only to develop plants with increased
agronomic importance, but also to develop plant cell cultures of importance to the
pharmaceutical industry. It will be appreciated from the discussion above, that any
reduction in the amount of land needed for growing plants for pharmaceutical purposes
will be important. Zenk and co-workers6 in a classical study carried out using cultures
derived from periwinkle (Catharanthus roseus) showed that it was possible not only to get
plant cell cultures to produce the pharmaceutically important indole alkaloids ajmalicine
and serpentine, but also to get cultures that produced these compounds in yields that on a
dry weight basis greatly exceeded the yield from whole plants. 6 This particular study was
extremely useful since it illustrated that the use of mutagens was not necessary to get cell
cultures that produce high yields of a desired metabolite. All that was utilized were
efficient alkaloid detection and cell selection systems, to recognize somaclonal variants
with the desired properties. In addition, however, these workers also carried out
pioneering studies on the role of plant hormones in influencing the production of
secondary metabolites. They showed that the type of plant hormone included in the
growth medium is of paramount importance for getting the expression of significant
amounts of secondary products by the plant cells growing in culture. Thus, while 2,4-
dichlorophenoxyacetic acid (2,4D) gave very good cell growth, the yield of alkaloids
obtained from the cells was very poor. By contrast, hormones such as indole-3-acetic acid
(IAA) gave good cell yields and encouraged alkaloid production by the cells. This led to

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the manufacture of an alkaloid production medium, and the concept that Catharanthus
cell cultures could be grown as rapidly growing suspensions in normal medium (containing
2,4D), until alkaloid production was required, at which point they could be transferred to
the special production medium.

Protoplast Fusion A second method with the potential for producing plants with new and novel
characteristics, is protoplast fusion. This technique involves dissolving away the tough
walls of plant cells with enzymes such as cellulase and pectinase, to liberate the plant cell
surrounded only by the plasma membrane (Fig 3). This process must be carried out in
media with suitable osmotic strength and ionic characteristics.l"2 If protoplasts of two
different plant species that do not normally interbreed are mixed, they can be induced to
fuse and form a heterokaryon by the addition of a 'fusagenic agent' such as polyethylene
glycol (PEG). Eventually the two nuclei may fuse to form a synkaryon. If the fusion
product can now be induced to divide and differentiate, a new plant species may result.
Originally this technique was thought to hold great promise for the development of new
plant species. However, it has been realized that there are a number of problems inherent
in the method. To mention some of the main problems; recognition and selection of the
fusion product from a mixture of similar parent protoplasts; regeneration of plants from
synkaryocytes; the plants so produced can possess a large number of undesirable qualities
such as sterility and disease susceptibility. 7 Thus, it appears to have become generally
recognized that rather than try to incorporate either a complete or a partially complete
new genome into any one plant cell (which is what protoplast fusion initially tried to
accomplish), it is much more logical to incorporate certain specific desired properties such
as herbicide resistance, which can often be localized to a specific gene, by using a small
piece of DNA containing the gene. Thus plant biotechnology has entered the era of
specific gene engineering.

Leaf materials or Cultured cells

1 Remove .
ui=ne'slioo
s w,,.[
enzymesolution
Surface ~
sterilization [~L=i=/~]
~ r ~ Protoplast
isolation
i~J.d~~ 60-80/Jm sieve
Protoplasts i -
~r Suspendin [ I i
Washing culture medium [ncubation
/

Droplets~50- 200/~1
T
Nucleus
Vacuole
Isolated
protoplast
Figure 3 Preparation and culture of protoplasts (redrawn from reference 1)
Genetic Engineering of Methods Which have been used in an attempt to genetically engineer plant cells, or are
Plant Cells actively being utilized at present, include transformation with isolated DNA, and the use
of specific vectors such as Cauliflower Mosaic Virus (CaMV), and species of Agrobac-
terium such as A tumefaciens and A rhizogenes.
Attempts to transform plant cells with DNA, have usually involved incubating either
cells or protoplasts with isolated DNA or RNA. A variation of this is the inclusion of the
nucleic acid in liposomes, 8 and the incubation of the liposomes with the plant cells. This
latter technique does appear to get round the problem of nuclease degradation of the

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nucleic acids to a limited extent. Success with this technique is rather limited by the fact
that the recipient cell must incorporate a large amount of DNA in what is essentially a
non-directed random fashion. The use of specific vectors greatly enhances the potential of
getting genetic information into plant cells and having it reproduced. Such vectors will
either have the ability to replicate and spread in a systemic manner throughout the plant
(CaMV), or else incorporate a particular piece of DNA into the plant genome, where it
will then be replicated in a Mendelian way. This objective could be accomplished using
Agrobacterium.
Cauliflower Mosaic Virus has been cited as a potential vehicle for introducing foreign
DNA into plant cells. 9 One of the principle attractions for using CaMV is the ease of
infecting plants with this agent~ accomplished by simply rubbing leaves with extracted DNA
plus an abrasive. The viral DNA is about 8kb in size, 9 and is in double-stranded form.
The small size of the viral genome places limits on the size of any foreign DNA that can be
inserted into it leaving the DNA infective and capable of being encapsuled into a normal
viral particle. Most experiments so far carried out have apparently utilized very small
(8bp) oligonucleotide insertions in various regions of the genome, with subsequent tests
for infectivity. 9
As already noted above, CaMV DNA does not appear to integrate into the plant
genome. Because of this, the viral DNA will be propagated in virion form throughout the
plant. This type of system could, however, be effective if the viral DNA is highly amplified
in plant cells, thus permitting the inserted DNA to code for large amounts of a foreign
gene product in the plant. By contrast to the CaMV system Agrobacterium species such as
A tumefaciens are able to integrate a piece of foreign DNA into the genome of a plant cell
where it is subsequently replicated in a Mendelian fashion in regenerated plants.t° A
tumefaciens is a gram-negative bacterium responsible for the production of tumerous
growths called crown galls in dicotyledenous plants. While Agrobacterium species enter
plants through a wound, they do not invade healthy cells. Virulent bacteria are able, via a
plasmid called the Ti-plasmid, to induce healthy cells to enter an unregulated growth
pattern and thus produce the gall. Tissue taken from this gall has two important
characteristics which are displayed in tissue cultures. Firstly, growth in cell culture
medium is hormone-independent (hence the unregulated growth of the gall tissue).
Secondly, the cells are able to produce amino acids called opines, which are entirely new
derivatives of familiar amino acids such as arginine, lysine, or histidine. Each tumour
produces one particular type of opine, depending upon the particular strain of
Agrobacterium used for infection. In order to become hormone-independent, and produce
opines, plant cells must be transformed by having a piece of the Ti-plasmid DNA
physically integrated into their own genome. Exactly how the Agrobacterium gets the
piece of DNA (called T-DNA) into the plant cell and integrated into the genome is not
known. The size of the T-DNA varies, from about 14kb to about 25 k b . 9
In recent years, a number of workers have successfully engineered Ti-plasmids to
contain selected genetic material, and then re-inserted the Ti-plasmid into Agrobacterium.
The engineered bacterium can then be utilized to infect plants or plant cells in culture with
expression of the inserted information. A very readable account of Agrobacterium and the
complex shuttle plasmid systems used in engineering the Ti-plasmid is provided by
Chilton.~l One of the most recent success stories that have shown the potential for the use
of Agrobacterium, is the transfer of the gene coding for the storage protein phaseolin,
from bean tissue (Phaseolus vulgaris) to sunflower tissue, where it is expressed.12

Future Potential of This review has stressed some examples of the future use of plants in biotechnology. While
Plants in Industrial it has tended to emphasize agricultural uses, it should be realized that Japanese workers
Biotechnology have already obtained many patents based on the pharmaceutical uses of plant cell
cultures. 13 Thus, in many senses it could be truly said that man's future health and welfare
may depend on the biotechnological uses of plant cells.

References 1 Plant tissue culture methods. (1982) Edited by L R Wetter and F Constabel, National Research Council of
Canada, Prairie Regional Laboratory, Saskatoon, Canada
2 Experiments in Plant Tissue Culture. (1982) Edited by J H Dodds and L W Roberts, Cambridge University
Press, London, England

B I O C H E M I C A L E D U C A T I O N 13(2) 1985