Factors Affecting

Morphogenesis In Vitro
1) Genotype ( Genetic
Composition)
2) Substrate- Media composition,
hormones, solid media & liquid
media, advantages of
solid/liquid media
3) Physical Factors of cultures/
Environmental Factors
4) Factors related to Explants
 Genotype
Culture medium & physical factors must be
optimum for a given sp. to grow well. Explants
differ in their responses/abilities to form organs.
Differ also in their abilities to withstand
sterilisation procedures. For callus culture,
ability/potential to form callus, growth rate
depend on varieties. For example in Arabidopsis
thaliana, only 3 out of 18 genotypes formed
callus.
The capability to form adventitious shoots also
different. Therefore need to use/screen many
different varieties in order to get the best
response.
 Substrate
Some sp. Can form shoots even in water.
Some need nutrients and sucrose and
some can only form shoots in certain ratio
of nutrients, sucrose etc.
The balance between hormones for shoot
formation or callus does not mean every
sp. Needs a special medium. Most
explants can form shoots and callus on MS
medium. The interaction between
endogenous and exogenous hormones is
important.
 Solid Medium
Advantages: easy for small explants
to be seen. Explants are placed on
the medium, easy for aeration. In
liquid medium need to be shaken for
aeration. Shoots and roots can grow
in a systematic manner in the solid
media. In liquid media, shoot buds
may formed but failed to form
shoots. In liquid media callus formed
may be broken up.
 Advantages of liquid Media
In liquid Faster growth due to larger
contact areas, more efficient in
nutrients uptake. If there is toxic
materials/substances can be diluted
in liquid medium. For eg. In orchid, 2
mm was obtained in 2 months as
compared with 4-6 months in solid
medium. Shoots ( Apple) grow faster
in liquid medium.
Cultural / Environmental Factors
Includes;

temperature
Humidity

Light

Container Size
 Temperature
The temperature experience by the cultures are
generally higher than the intact plants. Most cultures
are maintained at a constant temp. in the culture
room or incubators (day & night). Normally at 25C,
the range between 17-32C
Tropical & subtropical species (22.7C, range between
24-32C, depending on species & objectives of
experiments). The small change in temp. does not
affect growth/plant propagation so much. Eg
Streptocarpus, the optimum temp. 12C, when the
temp. increased to 30C, shoot formation decreased.
Potatoes formed 10 fold tubers at 20C compared to
at 27 or 28C.
In some cases 1-10C can be used to break dormancy.
 Humidity ( Moisture)
Important to prevent
dryness/dehydration in plants. The
relative humidity( RH) for in vitro
cultures, 70% or higher in culture
containers. Some spp need 80-90 %
RH. If less than 60%, the plants
normally failed to survive. Some
suggested that better to put more
than 1 shoot in a container to
prevent necrosis.
 Light
Light is necessary for photosynthesis.
Photomorphogenesis ( Development of
forms/structures) also need light although
in a small quantity.
Photosynthesis and Photomorphogenesis
depend on plant pigment which can absorb
light at a certain wavelength.
In Vitro photosynthesis is very low &
cultures depend on sucrose for nutrients.
Therefore light is more important for
photomorphogenesis.
Light Receptor in plants for red light =
Phytochrome, whereas for blue light is
Flavo- protein.
 Cont- Light
The light qualities which affect
morphogenesis are wavelength, flux
density, duration of exposure to light
or photoperiod. In cells/ tissue
cultures, light is not so important as
some species can divide in the dark.
 Container Size
Volume of containers can also affect
growth & morphogenesis in vitro. This
could be due to the concentration of
Carbon dioxide, ethylene and air spaces in
the culture containers. Bigger containers
can enhance growth, eg in tobacco pollen
culture, no. of embryos & plantlets formed
dependent on the container size. This
indicates that aeration is important in in
vitro morphogenesis.
 Factors Related to Explants
Depend on parts of the explants taken.
Treatment of the donor/parent plants

Physical of the explants.

These factors can interact with each other.

Cell determination, epigenetic changes

Parent/donor plants play a major role-

good materials produced good results.
 Explants
Type, size, age, culture conditions
- Influence morphogenesis and
regeneration

Age
i) Material age
ii) Phase of growth (mature, juvenile)
iii) Duration of culture, beginning from
the starting of cultures.
- All the above determine whether direct or
indirect morphogenesis.
- Phase of development and differentiation
also play a role although difficult to
determine.
- Eg. In Jasminium, only the shoot explant
can regenerate although other explants
can form callus.
- Callus from seedlings and inflorescence of
Cymbopogon have the abilities to
morphogenesis higher compared to callus
from seeds or roots etc.
- Sometimes it is difficult to pinpoint which
age factor that determine the expression
of morphogenesis.
 Culture Duration/Age
Loss of capability to form plantlets in some
callus can retain ability to form shoots for
a long time.
Some cultures can be maintained for 3-8
years.
Reasons for the loss of regeneration
potentials can be:

i) Genetic variation in cell population.

- Cells which can divide and multiply
rapidly will dominant the cultures, i.e cells
which can adapted well.
 - Therefore, if cells which are totipotent were not
the well-adapted, after sometimes the totipotent
cells will be lost.

ii) The presence of substances which can enhance

morphogenesis found in the explants.
- After sometimes this substance will disappear in
vitro. While the substances which can prevent
morphogenesis accumulated in cultures.

iii) Epigenetic changes

- Although genotype are always maintained
however, there are times when the mechanisms
regulating/ controlling information/ genetic
information may be not/ fail to function during
subculture.
- when chromosomal abnormalities occur and
accumulated in cultures potentials for
regeneration/ morphogenesis will decrease.
 Explant Size
Explant size will also determine the role
for regeneration. If the size is too small,
the explant wont survive in cultures. If
too big, difficult to sterilize.
Higher rate of contamination by bacteria,
fungi etc.
In Solanum laciniatum, explant (leaf)
smaller than 2mm, will not survive, but if
survive can produce number of shoots
equal to explant of 5 mm in size.
Big explant (10 mm) will produce good
results, the explants will lose contact with
the medium due to curling of the leaf
portions.
 Polarity in Regeneration
Most explants show polarity in
morphogenesis.
The top portion/ upper portion which has
potential for morphogenesis into shoots/
leaves known as distal, where as for roots
known as peroximal.
Stem explant normally form shoots from
the upper/ top part and roots from the
lower/ bottom portion.
Most leaves give rise to shoots and roots
from the bottom which is nearer to the
stem.
However, there are exception in some
cases.
 Root segments show a strong polarity. In
the root distal, floem cells divide rapidly to
form callus and subsequently adventitious
roots.
Callus formation in the root peroximal
ends is rare.
Natural polarity is due to the movement of
substances (growth) especially
transportation of IAA.
In small segments of tissues, polarity is
not obvious. The position of explants in
the media also influence morphogenesis.
Competition between Meristems
The first meristem that formed in cultures
may inhibit formation of other meristems.
Usually the first meristem will produce
substance, possibly auxin to prevent the
formation of subsequent meristem near
the first one.
Sometimes when the shoot buds are
removed, other shoot buds will form.
Competition is also found in somatic
embryo formation. If one piece of large
tissue is cultured/ subcultured, only 2-3
plantlets will form but if the tissues are
broken into 4-5 smaller portions, every
piece will form 1 plantlet.