Plant cell suspension cultures and production of secondary metabolites.

Plant cell suspension culture consists of a culture of single cell or small aggugatis of cells multiply while
suspended in agitated medium. It is normally initiated by transferring précis of undifferentiated and fable
caliber to a liquid medium which is tanutuely agitated by mechanical means. However in only the rarest of
unstanes, if ever, does such a culture exist. Increased cell disconcertion mean increased culture uniformity and
consequently most researches strive to achieve a five cell suspension as possible. However some degree of cell
aggregation generally have to be tolerated and so called fine suspension cultures consists of micro to sub-
macroscopic colonies made up of 5-200 cells. Further, culture consists of large aggregates (0.5-1.0mm in
diameter) usually are more attainable.

However in certain instances a degree of cell aggregation has been argued to be greatly beneficial-e.g.
in retaining the tolipotent character of gramineous suspension cultures and in obtaining enhanced yields of
desirable secondary metabolites from invitro system.

The mostly commonly used cell suspension is of two types:

1. Batch or closed type

2. Continuous culture

Batch Culture: - These are used for initiating single cell culture. Culture suspensions are grown in 100-250 ml
flasks, each containing 20-75 ml of culture medium. The cultures are continuously propagated by routinely
taking a small aliquot of the suspension cultures increases due to cell division and cell enlargement. This
continues for a limited period after which the growth stops due to exhaustance of some factor or the
accumulation of certain toxic metabolites in the culture medium. If at this stage, a small aliquot of the cell
suspension with uniformity dispensed cells and cell aggregates is transferred to a fresh method of the same
composition (sub-culture), the cell growth is revived. The biomass growth in batch cultures follows a fixed
pattern shown in fig.4.1

Fig.4.1

Initially the culture passes through a lag phase, followed by a brief exponential growth phase during which
active cell division occur. After three to four cell generation the growth declines and finally the culture enters
stationary phase. The duration of the lag phase would largely depend on the growth phase of the stock culture
at the time of subculture and the size of the inoculums (Stuart & street 1969).

Cultures cube maintained continuously in exponential phase by frequent (every 2-3 days) subculture of the
suspensions prolonged maintenance of the cultures in the stationary phase may result in extensive death and
lyses of the cells. It is therefore culture that suspensions are sub cultured soon after they have reached their
maximum day into yield (Street 1977). Addition of conditioned medium (in which cell cultures have been
grown before) reduces the lag phase dramatically (Bergmann 1977).

For sub culture of the suspension cultures a pipette or syringe with an orifice fine enough to allow single cell
and small aggregates (2-4 cells) to pass through is used. At the time of subculture the flask is allowed to stand
for a few seconds to allow the large colonies to settle down and the suspension is taken from the upper part of
the culture.

The culture of a callus is generally controlled and it may be difficult to obtain a good dispersion of
cells under any condition. However it has been possible to improve the tissue dissolution by manipulating the
media composition and subculture routine.

Addition of 2, 4-D (Negrutiu and Jacobs 1977) , small amount of hydrolytic enzymes such as
cellulose and pectin’s (Street 1977) or substances like yeast extract (Noguchital 1977), had a promotry effect
on cell dispersion (Negrutiv and Jacobs 1977) reported that maximum dissouation of cells was achieved by
maintaining the culture in the late log phase by adding fresh medium every day in a proportion that the
biomass/medium volume was kept at 2. As mentioning earlier friability of the callus tissue increases if it is
maintained on a semi solid medium for 2-3 passages.

However it must be borne in mind that even the first cell suspension culture is comprised exclusively
of free cells. Owing to certain inherent draw backs of the system, batch cultures are not ideal for studying of
cell growth and metabolism (Fowler 1977), (Street 1977),(Wilson 1980). Batch cultures are characterized by a
constant change in the pattern of cell growth and metabolism and the composition of nutrient medium. In
these cultures the exponential growth with a consistency of cell doubling time may be achieved for a short
time but there is no period of steady state growth in which the relative comentrations of metabolism and
enzymes are constant (Wilson 1980) .To some extent these problems are overcome by continuous culture.

Continuous Culture: A number of culture vessels have been designed to grow large-scale culture under steady
state , for long periods by adding fresh medium and draining out the used medium (Street 1977).Continuous
culture may be of the close type or the open type. In the former, the addition of fresh medium is balanced by
out flow of the old medium. Thus in close continuous cultures cell biomass continuous to increase as the
growth proceeds. In constant in the open continuous cultures, the inflow of the medium is accompanied by a
balancing harvest of an equal volume of the culture (medium and cells). The rate of inflow of medium and
culture harvest are so adjusted that the cultures are maintained at a constant sub maximal growth rate
indefinitely (Flower 1977). Two types of open continuous cultures have been used chemo stat and turbido
stat. In chemo stat cultures, a steady state of cell growth is maintained by a constant inflow of the fresh
medium in which the concentrations of a chosen nutrient (nitrogen, phosphorus or glucose) has been adjusted
so as to be growth limiting. In such a medium all constituents other than the growth limiting nutrients are
present at concentrating higher than the required to maintain the deserved rate of cell growth. In turbid stat
cultures, the input of fresh medium is intermittent, controlled by an over ease in the turbidity of the culture
from cell growth. A preselected biomass density is maintained by the washout of the cells.

Chemostat cultures offer the possibility of maintaining a steady state of cell growth and
metabolism and determine the effect of individual growth limiting nutrients as cell growth.
 However, despite the clear cut advantages continuous culture are not used widely for large scale
plant cell culture, probably because these cultures require a lot of attention and the equipment is not
generally available ( Wilson 1980).

Culture medium for suspensions:

The medium used for raising fast growing friable callus should generally prove suitable for initiating
suspension cultures of that species. Manipulation of the auxim /cytoicum ratio to achieve better cell
suspension is desirable, of course, agar is omitted from it.

For tobacco, increasingly the concentration of 2, 4-D from 0.3mgl -1-2mg-1 and supplementing the callus
medium with additional vitamin and casein hydrolysate herben recommended (Reynolds and Muraslugc
1979). In actively growing suspension cultures the inorganic phosphate rapidly utilized and consequently, it
soon becomes a limiting factor (Eriksson 1965; Noguchietal 1977) when phosphate concentration in the
medium was raised three times the original level, it was completely utilized by the cells within 5 days.

Agitation of the medium:

To achieve movement of the cultures in suspension cultures various types of setups have been used.
Muir(1953) who for the first time demonstrated that cells of tobacco and tagetes erecta ceube cultured in
suspension cultures, used an orbital platform with clips of appropriate size for holding the flasks, often the
clips are inter changeable to permit the use of flasks of different sizes. An orbital shaker with a variable speed
of 30-150 rev.min-1 is satisfactory speed above rev.min-1 is unsuitable; Street (1977) with stroke in the range
of 2-3 cm orbital motion.

Synchronization:

A synchronous culture is one in which the majority of cells proceed through each cell cycle
phase (G1, S, G2 & M) simultaneously. The degree of synchrony is expressed as percentage synchrony. For
studying cell cycle and cell metabolism. In suspension culture it is advantageous to use synchronous or
partially synchronous cultures which exhibit amplificatic of each event of the cell cycle as compared to non-
synchronous cultures (King 1980) .Since cell suspension cultures are normally a synchronous, considerable
efforts have been made to achieve a reasonable synchrony. Synchronization of a synchronous culture is
characterized by sequential alteration in frequency distribution of different cell cycle events. It has been
emphasis by King & Street (1977) and King (1980) that the degree of synchrony should not be determined
solely by meiotic index ; it depends upon number of parameters determined independently e.g.

a) The percentage of cells at a specific point in the cycle at one moment in time.
b) The percentage of cells passing a specific point in the cycle during a brief specific period.
c) The percentage of total time required for all the cells to plan a specific point in the cycle.

The methods to achieve synchronization of cell suspension fall under two catagones i.e. Starvation and
inhibition.
Starvation: In this method cells are first anertd in the G1 or G2 phase of the cell cycle by starring them of a
nutrient or hormonal factor required for cell division. After a period of starvation when the limiting factor is
supplied into a medium, the stationary cells enter division synchronously.

Up to 80-90% of the cells in the explants of tuber tissue of Helianthus tuberosus excised in low antenssily
green light and cultured in the dark on a nutrient medium containing 2, 4-D divided synchronously (Mitchell
1967;Yeoman & Evans 1967; Davidson & Yeoman 1974).

Suspension cultures of Accr psevdoplatanus from the stationary phase entered synchronous division when
they were inoculated in to fresh medium at low density (Street 1968).

Komamine etal (1978) achieved synchrony in vinca rasea cultures by starving them of phosphate for 4 day and
then transferring to phosphate-containing medium.

Growth hormonal starvation of cells has been used to synchronize cell cultures of Nicotiana tabacum var.
Wisconsin 38 (Cytokiven; Jouanneau 1971; Peaud-lenoel & Jouanneau 1971).

Inhibition: Inhibitions of DNA synthesis such as 5- aminouracit (Erikson 1966; Matting ley 1966; Kovacs & Vant
Hof 1970) hydroxy Urea and thymidine (Eriksson 1966) have been used to synchronize cell cultures. When
cells are treated with these chemicals the cell cycle proceeds only up to the G1 phase and the cells are
collected at the G1/S boundary. Removal of the culubitor is followed by synchronous division of cells.
However, in all these cases the only evidence of synchrony produced was fluctuation in the mitotic index
which is not entirely satisfactory.

Assessment of growth in suspension cultures growth in plant cell suspension cultures is commonly measured
by cell counting, determination of total cell volume (Packed cell volume, PVC) and fresh and dry weight
increase of cells and cell colonies.

1) Cell counting: Since suspension cultures are invariably cell colonies of various sizes. It is difficult to
make a reliable counting of cell numbers by taking samples directly from the flask. The accumany of
cell counting may be improved if the cells and cell aggregates are first dispersed by treating them with
chromic acid (5-8%) or pectinase (0.25%) .The method followed by street and coworkers, 1977 to count
sycamore cells is as follows: add 1 volume of culture to 2 volumes of 81. Chromic trioxide and heat to
70° c for2-15 minutes, the duration is determined by the growth of the culture, the mixture and shake
vigorously for 10 minutes before counting the cells in a haemocytometer.
2) Packed cell volume (PCV) : To determine packed cell volume, transfer a known volume of
unfortunately dispersed suspension to a 15 ml graduated centrifuge tube and spin at 200 g for 5
minute. PVC is expressed as ml pellet ml¯1culture.
3) Cell fresh weight (FW) : This caube determined by collecting cells on a pre weighed (in wet condition)
circular filter of nylon fabric supported in a Hantley funnel, washing the cells with water to remove the
medium draining under vacuum and reweighting.
4) Cell dry weight (DW) : Follow as above using a pre weighed dry nylon filter and after collecting the
cells on the filter, dry them for 12 hrs. at 60°c and reweight cell weight is expressed as per culture or
per 106 cells.
 5) Non-invasive methods : Recently two non-invasive methods to characterize growth in batch cultures
have been described. These are in addiction to above four methods were withdrawal of culture
samples is prerequisite.

Schripsema etal (1990) observed a direct correlation between loss of weight of

contents of a culture vessel and curve representing dissimulation of sugars into Co2. The dissimulation curve
clearly shows the different growth phases viz lag phase, exponential phase and stationary phase. Therefore
periodic weighing of cultural flasks provided with a stable closure for accurate correlation of water loss, a
dissimulation curve can be prepared and the growth characterized.

Assessment of viability of cultured cells :

There are four methods for determing the viability of cultured cells.

1) Phase contrast microscopy : Microscopic assessment of cell viability is based on cytoplasmic streaming
and the presence of a healthy nucleus (Negrutiu and Jacobs, 1977)
2) Reduction of tetrazolium salts: In the test , the respiratory efficiency of cells is measured by reduction
of 2, 3, 5 – triphenyl tetrazolium chloride (TTC) to the red dye formazan. Formazan can be extracted
and measured spectro photo metrically. Although this method allows quantification of observations. It
alone may not give a reliable picture of the cell viability ( Withers 1980).
3) Fluorescein diacetate (FDA) method : This technique offers a quick visual assessment of percentage
viability of cells. Stock solution of FDA at a concentration of 0.5% in acetone and stored at 0°c. To test
viability it is added to the cell or protoplast suspension for protoplasto , an appropriate osmotic
stabilizer is added to FDA solution at a final concentration of 0.01%.
After about 5 minutes , incubation the cells are examined, using a mercury vapour lamp with a suitable
excitation filter and suppress filter. FDA is non-fluorescing and non-polar and freely permeable across
the plasma membrane inside the living cell it is cleared by esterase activity releasing. The fluorescent
polar portion fluorescein since fluorescein is not freely permeable across the plasma membrane , it
accumulates mainly in the cytoplasm of intact cells, but in dead and broken cells it is lost. When
illuminated with UV light it gives green fluorescence (Widholm , 1972, Larkin 1976).
4) Evan’s blue staining : The stain caube used complementary to FDA. When the cells are treated with a
dilute (0.025%), solution of Evan’s blue the damaged cells take up the stain but the intact and the
viable cells exclude it and thus remain unstained.

Somaclonal variation
Somaclonal variation is defined as genetic or epigenetic changes that arise in- vitro between clonal
regenerants and their corresponding donor plants. The genetic changes are cytogenetic abnormalities
and alterations to specific sequences of DNA; epigenetic changes are alterations of gene expression
without changes to DNA sequences. Somaclonal variation, independent from the mechanisms involved,
has been reported for a number of plant species. The occurrence of somaclonal variation in tissue
culture has a negative effect on the rapid production of clonal plants of elite cultivars, but may promote
the production of novel horticultural crop genotypes. An improved understanding of the mechanisms of
variation in tissue culture, specifically epigenetic variation, may be a potential tool in producing
cultivars adapted to meet the growing demand for food.

One important advantage of tissue culture or clonal propagation in agriculture is the production of
uniform crops within a clone population. However, genetic variation can occur in these plants. The
term used to define the variation derived from any form of the cell or tissue culture is known as
“somaclonal variation”. The genetic variation can occur in isolated protoplasts, undifferentiated cells,
calli, and tissues of plants generate under in vitro conditions or tissue culture. The term “somaclonal
variation” was first introduced by two scientists Larkin and Scowkraft in 1981.

Until the first report of genetic variation in sugarcane reported in 1971, tissue cultures have been
extensively used to grow many horticulture plants, such as papaya, strawberry, banana, cucumber,
tomato, and citrus. However, after the incidence, the variation has been noted in many different crops.
And, in terms of commercial horticulture, somaclonal variations are considered to be worthless and
obstructive. These variations can cause a loss of genetic fidelity in crops. An important source of such
variation is chromosomal rearrangements, which can result in both genotypic (at the genetic level) and
phenotypic (at morphological or appearance level) differences. The variations at the genetic level can
be either due to the alterations in the chromosome structure (translocations, deletions, insertions, and
duplications), chromosome numbers (polyploidy and aneuploidy), or DNA sequence (base mutations).

Mainly, the somaclonal variations arise from the mutations that occur during tissue culture. The
mutations can occur due to variant stress factors including, hormonal imbalance in culture media,
exposure of tissues to chemicals during surface sterilization, and wounding. In many cases, oxidative
stress was found to be the main cause of somaclonal variation in tissue culture plants.

1. Explant Source

The genetic stability and fidelity of explants differ based on their source. The undifferentiated tissues or
meristematic tissues, such as cambium, procambium, and pericycle explants reduce variations.
However, highly differentiated tissues, such as stems, roots, and leaves, involve the intermediary callus
phase and thus produce more variations.

The types of explant chosen for the tissue culture procedure can affect the nature and frequency of
somaclonal variations.
Furthermore, the variation can also get inherited from the donor plants to your clones. If explants are
collected and prepared from a single donor plant, it increases the possibility of the occurrence of
somaclonal variation in tissue culture plants.

2. Media Components

Plant hormones are considered one of the major causes of somaclonal variation. Studies have reported
that the unbalanced hormone ratio in tissue culture media can introduce polyploidy in plants. Whereas,
their absence and low concentration use can result in producing normal plants. For example, adding
auxins to the culture media of calli or cell suspension can result in somaclonal variation induction due
to an increased rate of DNA methylation. Besides, the concentration of hormones, the type of PGR
(plant growth regulator) used in tissue culture can also cause somaclonal variation in tissue culture
plants.

3. Effect of Genotype

The genotype of the plants plays a major role in inducing somaclonal variations. And, they are highly
affected by the situation around them. Thus, stress can also cause variations in plants. However, plants
with different genomes respond differently to a variety of stress conditions. Thus, the genetic makeup
of the plant has a key role in the stability of the plant during tissue culture—where some genomic
components help plants to prevent stress and variations, some others make them unstable.

4. Regeneration Systems

Cellular organization is one of the essential factors in terms of plant growth. And, the loss of cellular
control can give rise to somaclonal variations in tissue culture. The regeneration systems (explants used
to regenerate plants) in terms of genetic stability can be arranged in descending order as:
micropropagation by preformed structures (such as shoot tips or nodal explants) > adventitiously
derived shoots > somatic embryogenesis > organogenesis from callus, cell, and protoplast cultures.
The regeneration of plants skipping the intermediate callus formation stage can be a way to reduce
variations in cultures. Techniques like somatic embryogenesis and axillary branching have also been
seen to provide promising results in reducing somaclonal variations. Therefore, there are extensively
used in commercial space. However, these techniques also do not offer 100% elimination of variations,
as some studies report.

5. Duration and Number of culture cycles

The number of subcultures and their duration is directly proportional to the introduction of somaclonal
variations in tissue culture plants. For example, an increase in the number and duration of subcultures
can cause an increased rate of somaclonal variations in plants. Moreover, rapid multiplication of plant
tissues in cultures can also cause instability and the plant genome and induce somaclonal variations.
Such cases have been observed during tissue culture of wheat (during a long period of subculturing)
and in micropropagated bananas during progressive subculturing.

Advantages and Disadvantages of Somaclonal Variations

Advantages of Somaclonal Variations

 Somaclonal variation has a major role in crop improvement.

 The variation can be a useful approach to introducing new varieties in plants, especially in the
case of growing ornamental plant species.

 It can be a useful approach to breeding new species.

 Somaclonal variation can be used in introducing specific characteristics in plants, such as

resistance to a spectrum of diseases, pathotoxins, herbicides, and biotic and abiotic stresses.

 It can also be used to increase the production of secondary metabolites.

Disadvantages of Somaclonal Variations

 It can cause the introduction of some undesirable characteristics in plants.

 The variants can be produced randomly and thus can be genetically unstable.

 It requires multiple rounds of field trials.

 It can produce undesirable results.

 The variants developed through somaclonal variation can have a pleitropic effect—one variation
can affect more than one characteristic and feature in plants.

 The variation is not used for complex agronomic traits, such as yield and quality of plants.
How To Identify Somaclonal Variation

Somaclonal variations in plants can be identified at four levels:

1. Morphological Level: Variations can be identified at the level of appearances, such as differences in
plant height, abnormal pigmentation, and leaf shape and size. For example, the stature of plants, tall
plants, and dwarf plants, can always be easily identified when grown. However, these differences can
also be induced due to changes in environmental factors. Thus, for the commercial production of
plants, the technique is not considered an effective approach.

2. Physiological Level: Physiological responses are reactions of plants to certain stimuli. Analyzing
plants at a physiological level for the presence of variations offers an effective approach to identifying
the variants faster at the young stage ad reduces overall loss. Some examples of criteria that can be
used for physiological monitoring include disturbances in gibberellic acid metabolism and level,
photosynthetic activity, and pigment synthesis in plants.

3. Molecular Level: Changes in the plants at the molecular level include changes in the chromosome
structure and number. Though, if present at a low concentration, these changes appear at the physical
level. However, when the alterations are at a high level, molecular studies like restriction fragment
length polymorphism (RFLP) are used to identify the variations.

4. Cytological Level: Observing variations at the molecular level can be a tedious process. Thus,
techniques like flow cytometry are used to count and examine chromosomes. The technique is
convenient and rapid compared to other available techniques to identify somaclonal variations in tissue
culture plants.