Hyperhydricity, Hardening, Packaging and transport, Qualtity control, Plant tissue culture

1. Advances in
Plant Tissue Culture
Jaskaran Kaur Arora
L-2016-A-19-D
Biotech 606

2. CONTENTS
Advances in overcoming problems
o Hyperhydricity
o Contamination
o Phenolic exudates
o Habituation
Hardening
Packing & Transportation
Quarantine for tissue culture raised plants
Quality control

3. Hyperhydricity
Hyperhydricity (vitrification) is a physiological malformation that results in excessive
hydration, low lignification, impaired stomatal function and reduced mechanical
strength of tissue culture-generated plants.
Hyperhydric plantlets remain stunted,
and show a typical ‘glassy’ appearance
Thick, translucent, curled, and brittle
leaves
These plantlets survive very poorly

4. Effects (Considerable losses in:)
 Commercial micropropagation industry
 Conservation of plant resources and genetic transformation of
plants

5. CAUSES
High salt concentration and High relative humidity
Low light intensity
Ethylene, a stress-related plant hormone accumulation
Concentration and type of gelling agent
High ammonium concentration
The conc. of microelement and hormonal imbalances

6. Control
• By modifying the atmosphere of the culture vessels
Adjusting the relative humidity in the vessel
Use of gas-permeable membranes (increased exchange of water vapor and
other gases such as ethylene)
Using higher concentration of a gelling agent, higher-strength gelling agent
HH can also be controlled by bottom cooling, which allows water to condense
on the medium
• Decreasing the ratio of NH4
+:NO3
- in the medium
• It was also shown that an increase in calcium in the medium reduced
hyperhydricity
• Silver ion (Ag+) inhibits the activity of ethylene
For more details, Read: https://sci-hub.tw/10.1016/b978-0-12-802221-4.00013-3

7.  About 67% of the hyperhydric plantlets
were found to revert to normal condition
when the plantlets were cultured in
medium supplemented with 29.4 mol/L
AgNO3
 Water content and hydrogen peroxide
(H2O2) content in the guard cells of these
plantlets were reduced
 AgNO3 also reduced the content of
endogenous ethylene
 Provided clues for exploring the cause of
excessive water accumulation in
hyperhydric plants

8. CONTAMINATION
SURFACE contamination: surface
sterilization
INTERNAL contaminants: Antibiotics,
Thermotherapy, Culture of explants free
of organisms , Maintenance of asepsis,
(Procedures are carried out in sterile
laminar flow hood)

9. PPMTM - Plant Preservative Mixture
• Plant culture preservative and broad-spectrum biocide
• Penetrate the fungus or the bacterium cell wall and inhibit the activity of key
enzymes in the metabolic cycles such as the citric acid cycle and the electron
transport chain
• Also inhibit the transport of monosaccharides and amino acids from the medium
into the fungus or bacterium cells
• PPM™ is less expensive than antibiotics, making it affordable for wide and routine
use.
• Since PPM™ targets and inhibits multiple enzymes, the formation of resistant
mutants towards PPM™is unlikely.
• PPM™ is heat stable and in general can be autoclaved with media.
ADVANTAGES OVER ANTIBIOTICS

10. Application of CuSO4 at 5 and 10 μM is helpful in controlling endogenous contamination and
promoting growth in E. variegata in vitro cultures
Higher concentrations were toxic and caused decrease in chlorophyll content along with
overall growth of the cultures.
Optimized the concentration of the metal in the growth media to tackle endogenous
infection while also increasing the growth of the cultures, unlike the antibiotics, which
inhibits plant’s growth.
Use of Cu for suppression of
microbial contamination

11. 10.0 μM 20.0 μM

12. Phenolic Exudation
The exudates are phenolic compounds that oxidize to form a brown material in the
medium that tends to be inhibitory to development.
Various treatments to minimize this
problem
Treating the explant with an
antioxidant (citric or ascorbic acid)
Including an adsorbent material in the
medium (polyvinylpyrrolidone or
activated charcoal)
Frequent transfers to a new
medium

13. • Genotype
• Explant age
• Light intensity
• Sterilization
• Put cultures to the dark in a fridge at about 5°C
• In cotton tissue culture, to reduce 'browning' caused by phenolic compounds,
glucose (instead of sucrose) as carbon source, and use 'Phytagel™' as gelling
agent (instead of agar)
Other factors

14. Tocopherols (or TCP) are a class of chemical compounds of which many have vitamin
E activity. Alpha-Tocopherol is the form of vitamin E

15. • In addition, cold pretreatment at 4°C for three or five days with or without
soaking in antioxidants were investigated
• Results:
1. Soaking of shoot tips in vitamin E before in vitro culture reduced browning
2. Keeping the explants for 3 days at 5°C decreased browning percentage
3. Vitamin E as well as cold pre-treatments enhanced callus induction
significantly

16. Pre-treatment with V.E at 40% is recommended to achieve the lowest browning and the
highest callus induction results in date palm cultures
Cold pre-treatment for three days at 5°C is recommended when the using of antioxidant is
not preferred

17. Phenolics
Phenylpropanoid Pathway
Phenylalanine Ammonia Lyase
(PAL)

18. Various levels of 2-aminoindane-2-phosphonic acid
(AIP)
Visual browning scores (1-10)
Cultured on 4.5 μM 2,4-D based media
2- aminoindane-2-phosphonic acid (AIP)
Competitive PAL inhibitor

19. Browning can also be reduced by:
• Choice of juvenile explants
• Culture in darkness
• Transfer to fresh medium at short intervals
• Culture in liquid medium
• Soaking explants in solutions containing antioxidants prior to
inoculation
• Choice of a low salt medium and proper GR

20. Habituation
Habituation is when a culture continues to develop in the absence of auxin or
cytokinin
Stable heritable loss in requirement of
growth factors by tissue culture plants
Shoot cultures habituated for
cytokinin continue to produce new
shoots on a cytokinin-free medium

21. • Proliferation of undifferentiated callus tissue and the formation of shoot structures
are all cytokinin-dependent processes.
• Habituation refers to a naturally occurring phenomenon whereby callus cultures,
upon continued passage, lose their requirement for cytokinin.
• Increased levels of expression of the cytokinin receptor CRE1 and altered levels of
other genes involved in cytokinin signaling
• Up-regulation of the transcription factor, transposon-related elements, and several
DNA- and chromatin-modifying enzymes which indicated that epigenetic changes
contribute to the acquisition of cytokinin habituation
Transcriptome-based analysis:
Introduction

22. Hardening
Transferring the
plantlets from in vitro
to ex-vitro; is a
crucial step in tissue
culture
Minimum survival
rates when cultured
plants re-established
Expose plants to
natural
conditions in step
wise manner
↓
High Humidity:
Fog
Mist
Clear cups
Hardening :
Controlled
Temp, light &
humidity

23. Gradual Adaptation to ex-vitro
• Usually the common method to acclimatized in vitro propagated plantlets
using weaning chamber (scheduled misting system)
• Several researchers have reported the use of:
 different potting media
 Bio-inoculants
 Propagation structures for proper hardening and successful
establishment in the open conditions

24. • The right potting media determines the success in acclimatization of
tissue cultured plantlets
• The most commonly used components:
 Peat
 Soil
 vermiculite
 organic composts and residues of the agroindustry like rice hulls,
Pinus and Acacia mearnsii bark

25. In the present study cocopeat showed
to be far superior to other potting media
95% survival
Better aeration, water holding
and nutrient supplying capacity of
cocopeat as compared to
vermicompost, sand and vermiculite

26. • Flats: shallow plastic, Styrofoam, wooden, or metal trays, with drainage holes
in the bottom
• Plastic Pots : nonporous, reusable, lightweight, and use little storage space
because they will nest
• Fiber Pots: pots are biodegradable, they are set in the soil along with the
plants
• Paper Pots : pots are biodegradable, and the seedling plug can be planted
intact into a larger container or into the ground without disturbing the root
system
CONTAINERS FOR PROPAGATING AND GROWING YOUNG
LINER PLANTS

27. Flats
Plastic pots
Fibre pots Paper pots

28. Packing & Transportation
All the hardened plants in poly bags are either dispatched in ventilated plastic
crates or cardboard cartons
The plants are removed from poly bags, washed free from soil, wrapped in tissue
paper, and packed in ventilated cardboard cartons
Each package is affixed with a certification label Duly signed; labels are provided by
the Accredited Test Laboratory (ATL) to the Tissue Culture Production Facility

29. Record of the deployment of the labels in terms of the size of packages,
number of plants per package, the name and contact details of the
consignee etc
A small handout or printed leaf let is placed on each package giving
relevant information about after-care of tissue culture plants
The packages carry appropriate instruction such as “HANDLE WITH CARE-
TISSUE CULTURE PLANTS”
. . .
For more details, visit: http://dbtncstcp.nic.in/Portals/0/Images/SOP-TCPF.pdf

30. LIST OF INSPECTION AUTHORITY
S.No. Name of Inspection Authority Jurisdiction Purpose
1 Head, Advance Centre for Plant
Virology, Indian Agricultural Research
Institute, PUSA, New Delhi
Entire Country Tissue culture raised
plants
2 Head, Indian Institute of Horticulture
Research, Hesarrgatta, Bangalore
Entire Country Tissue culture raised
plants
3
Head, Bio-resources Technology,
Palampur, Himachal Pradesh Entire Country
Tissue culture raised
plants
Plant Quarantine India
http://agritech.tnau.ac.in/crop_protection/crop_prot_quarantine_list%20of%20inspection%20authorities.html

31. Main features
No consignment of seeds/planting materials are imported into India
without a valid ‘Import Permit’
No consignment of seeds/planting materials are imported into India
unless accompained by a 'Phytosanitary Certificate'
All consignments imported into India through station, seaport, airport and
other entry points; are inspected and if necessary, fumigated, disinfected
by authorised plant quarantine officials

32. Materials requiring isolation are grown in post-entry quarantine facility
certified by the Designated Inspection Authority (DIA) to the conditions
laid down by the Plant Protection Adviser to the Govt. of India
Hay, straw or any other materials of plant origin shall not be used as
packing material
Import of soil, earth, sand, compost, and plant debris accompanying
seeds/planting materials is not permitted.
For more details, visit: http://www.nbpgr.ernet.in/Divisions_and_Units/Plant_Quarantine.aspx

33. 1. The tissue culture facility shall have small growth chamber or separate
incubation place for holding imported tissue.
2. The facility shall have an isolated poly/screen house facility for tissue culture
hardening.
3. The facility shall have Enzyme Linked Immuno-Sorbent Assay (ELISA) facility for
virus screening of stock material.
Requirements of tissue culture quarantine facility
http://plantquarantineindia.nic.in/pqispub/html/PostQReq.htm

34. a) Double door entry
b) The vents/openings shall be screened with insect-proof mesh of stainless
steel/phosphor bronze, meshes and woven wire. If nylon mesh is used it should be
of good quality and evenly woven
c) The drainage must be through a gully trap with a provision for insect-proofing in
case of concrete flooring
d) The entrance shall be provided with disinfectant pad for sterilization of feet and
basin for disinfecting hands
e) Suitable yellow sticky traps shall be hanged at various intervals at crop canopy level
to monitor insect population.
Some of the Requirements of closed quarantine facility
(glass/screen/poly house)

35. Quality control for tissue culture raised
plants
1. Virus indexing
Virus indexing is the testing of plants for the presence or absence of viruses. Each
meristem tip or callus or callus-derived plantlets are tested before using it as
mother plant to produce virus-free stock

36.  Genetic stability is of a major importance in micropropagation of any crop
species
 The assessment of the genetic stability of in vitro derived clones is an
essential step for micropropagation of true to type clones
 If the analysis is carried out in the initial stages, one may suitably
modify the micropropagation protocols to avoid the variation
2. Genetic stability
Causes of Variation:
Genotype of parent
Change in chromosome number
Change in chromosome structure
Point mutation

37. Approaches
To maintain the genetic constitution of a particular clone through its life span :
 Morphological observation
 Cytological analysis
 Molecular markers