2. Hardening:
The ultimate success of micropropagation on a commercial scale depends
on the ability to transfer plants out of culture on a large scale, at low cost
and with high survival rates.
During field transfer the in vitro grown plantlets are unable to compete
with soil microbes and to cope with the environmental conditions.
The in vitro culture conditions result in the plantlets with altered
morphology, anatomy and physiology.
In order to increase growth and reduce mortality in plantlets at the
acclimatisation stage, efforts are focused on the control of both physical
and chemical environment and bio-hardening of micropropagated
plantlets.
3. Continued….
cultured plants have non functional stomata, weak root system and poorly
developed cuticle
with an excess of phytohormones show abnormalities in morphology and
anatomy and are called vitrified plants
Acclimatization is carried against:;
Abiotic and
Biotic stresses
4. Light intensity:
In vitro growing plantlets are under low light intensity (1,200–3,000 lux)
and temperature (25 ± 2C), hence direct transfer to broad spectrum
sunlight (4,000–12,000 lux) and temperature (26–36C) might cause charring
of leaves and wilting of plantlets.
microshoots from in vitro to ex vitro conditions under direct sunlight
might cause photoinhibition and chlorophyll (Chl) photobleaching,
photoinhibition might be the cause of the transient decrease in
photosynthesis after transplantation.
Micropropagated plantlets can be left in shade for 3–6 days under diffused
natural light to make them adjust to the conditions of new environment.
This helps in semi-hardening of plants and leads to shoot elongation.
5. Humidity:
The retardation in development of cuticle, epicuticular waxes and
functional stomatal apparatus during in vitro culture cause high stomatal
and cuticular transpiration rates of leaves in plantlets when taken out of
the culture vessels.
In order to avoid this, slowly the plantlets should be transferred from high
humidity to low humidity conditions.
Microshoots should be kept in the shade with plugs loosened and after a
week or two, they should be transferred to pots containing sterile soil and
sand mixture covered with polybags.
Slowly stomatal and cuticular transpiration rates gradually decreases
because stomatal regulation of water loss becomes more effective and
cuticle and epicuticular waxes develop.
6. continued,…
Even if the water potential of the substrate is higher than the water
potential of media with saccharides, the plantlets may quickly wilt as water
loss of their leaves is not restricted.
In addition, water supply can be limiting because of poor conductivity of
roots and root-stem connections.
Many plantlets die during this period.
7. carbohydrate
Plantlets are developed within the culture vessels under low level of light,
on a medium containing ample sugar and nutrients to allow for
heterotrophic growth and in an atmosphere with high level of humidity.
carbohydrate concentration influences the acclimatization process because
plantlets switch from heterotrophic to autotrophic growth and any
treatment before and after transfer increases the photosynthetic capacity
of plants, which may improve plant establishment.
A sucrose concentration of 40 g/l added prior to transferring watercress
microcutting to in vivo conditions was shown to increase the dry weight of
established plantlets (Wainwright and Marsh 1986)
8. RETARDANTS
retardants can be used in micropropagation to reduce damage due to
wilting without deleterious side effects.
Use of paclobutrazol (0.5–4 mg/l) in the rooting medium is reported to
result in reduced stomatal apertures, increased epicuticular wax, shortened
stems and thickened roots, reduction in wilting after transfer to compost,
and also increased chlorophyll concentration per unit area of leaf.
9. ABSCISIC ACID
Abscisic acid (ABA), a naturally occurring plant hormone critical for plant
growth and development, plays an important role in plant water balance
and in the adaptation of plants to stress environments including low
temperature.
Hemavathi et al. (2010) found that ascorbic acid was expressed in
transgenic potato by over-expressing L-gulonoc-lactone oxidase (GLOase)
gene and accumulation of ascorbic acid showed tolerance to abiotic
stresses.
10. Biotic stress
Biotization:
Another major cause of high mortality of microshoots is their sudden exposure
(particularly the root system) to microbial communities present in the soil as they do
not possess sufficient resistance against the soil microflora.
Endophyte refers to the fungi and bacteria which invade or live inside the tissues of
plants without causing any diseases or injury to them. They also promote growth of
the host plant and the formation of secondary metabolites related to plant defense
(Hao et al. (2010).
In vitro co-culture of plant tissue explants with bacteria and vesicular arbuscular
mycorrhiza induces developmental and metabolic changes in the derived plantlets
which enhance their tolerance to abiotic and biotic stresses.
. By using rhizosphere bacteria, successful biohardening of tissue culture raised tea
plants have been reported by Pandey et al. (2000).
11. Cost effective method:
Deb and Imchen (2010) reported an alternative approach to acclimatize the
microshoots of orchids by using an alternate substratum.
In-vitro hardening using 10% MS medium, with no carbon source or any
any plant growth regulators.
Charcoal pieces, small brick chips, mosses, decay forest/wood litter in
different ratio.
