This document discusses the process of acclimatizing plantlets grown in vitro to ex vitro conditions. It defines acclimatization as the process where plants adapt anatomically, physiologically and morphologically to the new ex vitro environment. Plantlets grown in vitro develop deficiencies due to different growth conditions compared to ex vitro. The steps of acclimatization involve transferring plantlets to a humid, low light environment to harden off before transitioning them to higher light and lower humidity conditions in a greenhouse. Acclimatization is necessary for plantlets to regain tolerance capacities and survive transplantation outside the laboratory.

1. Acclimatization of Plantlets
March 11, 2016
Department of Biotechnology, PRSU, Raipur
By: Sajal Shrivastav
M.Sc II Semester
Guided by:
Dr. Afaque Qureshi
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2. • Introduction
• Transplantation and Acclimatization
• Difference in growth conditions : in vitro & ex
vitro
• Deficiencies of in-vitro grown plants
Morphological Deficiencies
Anatomical Deficiencies
Physiological Deficiencies
• Steps in Acclimatization
• In-vitro Acclimatization
• Significance of Acclimatization
• Conclusion
• Reference
Synopsis
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3. The ultimate purpose of plant tissue culture technology will not be
achieved if plantlets grown in vitro do not survive in harsh and
uncontrolled environment of the field.
This technique of transferring the lab grown plantlets into field is
TRANSPLANTATION or Stage IV.
Transplantation is the crucial and utmost care appealing stage.
Acclimatization is the principle that governs Transplantation.
INTRODUCTION
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4. Definition
“Acclimatization is the phenomenon whereby plants
morphologically, physiologically and anatomically adjusts from in-
vitro to ex-vitro environmental growth conditions.”
“It is the process during which plants become adapted and gets
accustomed to a new culture and environmental ambience.”
Acclimatization is also known as hardening off.
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5. Etymology of acclimatization :
• Root word is climate.
• Derived from ancient greek word “klima” means “inclination”
or “slope”.
Ac + climate+ ization.
• Acclimatization word is also used for other living systems.
Eg. Acclimatization at high altitudes
Acclimatization after heibernation.
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6. Transplantation & Acclimatization
It’s a technique/step in PTC.
Its inbuild ability of plants to
adapt/respond to changed
environment. 6

7. Difference of growth conditions : In-vitro & ex-vitro
In-vitro
• High conc. Of organic and
inorganic nutrients.
• Presence of Growth Regulators
• High Relative Humidity.
• Low Light Intensity.
• Low Temperature.
• Poor gaseous exchange.
• Utmost physical care is taken.
Ex-vitro
• Photoautotrophic nutrition
allowed only.
• No growth regulators.
• Low relative humidity.
• High light intensity.
• High Temperature
• Gaseous Exchange must be done.
• Chances of physical tragedy like
wind flow, water currents etc.
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8. Deficiencies in in-vitro grown plants
Deficiencies are due to the fact that in-vitro regeneration focuses
only at providing the optimal growth conditions required for the
explant to grow rapidly.
There are three types of deficiencies observed :
• Morphological Deficiencies
• Physiological Deficiencies
• Anatomical Deficiencies
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9. Morphological Deficiencies
Shoot
• Reduced length
• Reduced number
• Reduced dry mass
Root
• Reduced length
• No root hairs
• Reduced dry mass
Leaves
• Reduced blade
expansion
• Reduced number
• Reduced length
• Reduced internodal
length
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10. Anatomical deficiencies
• No/Reduced formation of cuticle and epicuticular wax.
• Thinner epidermal layer
• Thin Cell wall
• Reduced Sclerenchyma/Collenchyma formation
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11. Poor Anatomical differentiation in leaves
• Stomata - Greater stomatal frequency
Impaired stomatal growth
Light/dark insensitive stomata
• Mesophyll – Poor differentiation
Palisade is poorly developed
Loosely organized Spongy mesophyll (large intercellular spaces.)
• Weak Vasculature in leaves
• Chloroplast – poorly developed
Low chlorophyll contents
Disorganised grana
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12. Physiological deficiencies
Low light
High Humidity
Less CO2
High Sucrose
HETEROTROPHIC
NUTRITION
• NO photosynthesis
• Less Chlorophyll
• Inactive / No photosynthetic
enzymes
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13. Steps in Acclimatization
1. Transfer plantlet from in-vitro culture tubes.
2. Wash its roots to remove agar
3. Primary Hardening –
Potting mixture- contains no organic nutrition but low inorganic
nutrition. Eg : Peat, Perlite, polystyrene beads, vermiculite, fine bark,
coarse sand etc or their mixtures in different proportions are used.
External N, P, K and urea are also applied.
High Humidity (90-100%) and low light is provided in a misting/fogging
chamber for for 10-15 days. 13

14. 14

15. Methods to provide high to low humidity:
• Cover plants with transparent plastic bags with
small holes in it and increase decrease the
humidity gradually by enlarging those holes.
• Culture vial cap is loosened day by day (7-10 days)
to create humidity gradient. Last the vial is kept
uncapped. Then transplanted in potting mix.
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16. Misting and Fogging systems :
Sophisticated chambers providing Humidity gradient using saturated air.
Misting is cheaper but less efficient as causes nutrition to leach and making plants dry.
Fogging is expensive but has no such problems as misting. Produces water droplets less
than 20µm.
Image- Misting tunnel (droplet size more) Image- Fogging chamber (droplet size very less)
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17. Tropical countries uses this technique to create Humidity
Gradient:
One side has wet curtains of cellulose or polystyrene pads which are constantly
supplied with water on other side exaust fans.
This creates a Humidity gradient along with Temperature gradient (5-6 ˚C reduction)
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18. After that 4-6 weeks plantlets are transferred from High Humidity-low light
zone (of misting/fogging chamber) to Low humidity-High light zone(Green
house or nethouse).
Image – a green house Image – a net house
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19. Use of Anti-transpirant to reduce water loss during transpiration:
• Leaf-surface covering agents such as glycerol, paraffin and grease
promoted ex-vitro survival of several herbaceous species (Selvapandiyan
et al.)
• Stomatal transpiration of micro-propagated cauliflower plantlets were
reduced with a leaf spray of 10mM ABA (Wardle et al.)
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20. In-vitro acclimatization
Why its done?
• Ex-vitro acclimatization is labour
intensive
• High capital required
How its done?
• Infecting in-vitro plantlets with Rhizobium.
• Using culture vials with microporous lids.
• Use of dessicants on culture vials.
• Use of certain growth retardants ( anti
gibberellins ) shows more survivality in ex-
vitro as reported.
• Induction of storage organ formation by
manipulating sugar/PGR contents.
Image- Paclobutrazol effect on transplantation
Image- Micro-
porous culture
vials.
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21. Significance of Acclimatization
• Allows plants to regain its actual tolerant capacity.
• Necessary for higher survival rates of in-vitro plantlets.
• In-vitro acclimatization can help reduce the time
requiring criteria of ex-vitro hardening.
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22. Conclusion
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23. References
• Plant Tissue Culture Theory and Practice, S S Bhojwani
and M K Razdan.
• https://www.academia.edu/2514456/Acclimatization_of_
tissue_cultured_plantlets_from_laboratory_to_land
• http://www.iisc.ernet.in/currsci/dec252003/1704.pdf
• http://www.isaet.org/images/extraimages/P315405.pdf
• http://digitool.library.mcgill.ca/webclient/StreamGate?fol
der_id=0&dvs=1457636214994~299
• http://www.scielo.br/img/revistas/babt/v57n5//1516-
8913-babt-57-05-00636-gf01.jpg
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