1. Problems in tissue culture
Culture contamination
Vitrification
Acclimatization
Post culture behavior

2. Culture Contamination
Two sources:
Carry over of microorganism on the surface or in tissue of explants
Through faulty procedures in the laboratory

3. Culture Contamination
Cause economic losses, by overrunning the culture either killing the explant or rendering it for the subculture
Affect the productivity both in vitro and of the progeny plant

4. Organisms associated with plant surface
Fungi including yeast
Bacteria
Mollicutes (mycoplasmas, spiroplasmas and related organism)

5. Endophytic microorganisms
Intercellular endophytic microorganism
Virus
Viroid
Fastidius prokaryotes
Intracellular endophytic microorganism
L-forms of common plant associated bacteria
Fastidius bacteria associated with plant vascular tissue
vector transmitted and may be spread by contact between infected and healthy plant
they are capable of clonal propagation

6. Aspect of quality control
Awareness of the range and natural history of possible contaminant of the crop, including specific pathogen
Adequate preparation of the donor plant including treatment to reduce or eliminate pathogens
Confirmation of the status of culture in stage I following employment of strategies to obtain healthy cultures and again based on reliable screening methods

7. Aspect of quality control
Rigorous monitoring of production to confirm the status of the cultures. In large scale production this will necessitate sampling production and is dependent on an appropriate sampling protocol
An awareness that the spectrum of contaminating microorganism may alter with time in culture
Monitoring of progeny based on sampling of production

8. Detection and identification methods
Test
Application
Non specific tests:
Culture indexing
DNA staining
Leaf dip electron Microscope
Gel electrophoresis
Cultivable bacteria
Mycoplasmas and related prokaryotes
Viruses
Viroids
Specific tests:
ELISA
DNA probes
Rapid diagnostic kit
Fatty acid profiling
Viruses and bacteria
All organisms
Bacteria

9. Vitrification Translucency Hyperhydration Succulency Glassines
Change into a glassy appearance
Produce fragile plants which have a glassy and hyperhydrous appearance

10. Vitrification
The cells were surrounded by thin walls and contained a relatively poor and largely vacuolated cytoplasm
Increased cellular space due to extra protoplastic water
Many of the chloroplasts lacked of the normal organization into grana and stroma
Chloroplasts contained large starch grains whereas the chlorophyl content was lower
Defective epidermal tissue
Faulty deposition if epicuticular waxes
The guard cell did not function properly
Abnormal stomata
Reduced lignifications

11. Factors associated to vitrification
Physical and chemical state of the medium
Type of culture media
Gelling agents
Organic component
Inorganic component
Growth regulators
Relative humidity
Environmental condition
Various addition

12. Acclimatization
Process during which plants or other organisms become adjusted or accustomed to a new climate or situation as a result of natural processes
Hardening-off
Moving the complete plants to greenhouse or field
Not unique to micro-propagation

13. Why acclimatization is important?
The greenhouse and field have substantially
lower RH
higher light levels
autotrophic growth
septic environment
 stressful to micro-propagated plants compared to in vitro condition

14. Common issue Agar has to be thoroughly washed from the root
Pesticide may be phytotoxic to some species of micro-propagated plantlet
Gradually reduce the RH or amount of mist
Maintain light level in the greenhouse at 50% shade before plants are transplanted to the field

15. Acclimatization Control environment acclimatization
Specific place that all environmental conditions can be control either automatically or manually
Direct field acclimatization
Transferring the rooted plantlet directly to the field

16. Important aspect in the control environment acclimatization
Humidity
Light
Soil and container
Diseases
Temperature
Nutrient

17. Control humidity
Avoid the use of an automatic mist system due to mist leaches nutrients, causes the medium to become too wet, allows the plantlets to dry, creates an environment favorable for the growth of algae and some fungi and bacteria
Fogging
The use of a humidifier
Placing plantlets in an enclosed area that will water vapor
The use of anti-transpirants to reduce water losses

18. Light
While in vitro, plantlet has been exposed to relatively low level of light and their leaves are thin and thus resemble shade leaves
Leaves of plantlet place under too high a light level will become chlorotic and necrotic
Shading up to four weeks under up to 90% will reduce transpirational demand and excessive light that can destroy chlorophyll molecules
Following a period of shading, plantlet should be gradually moved to the light level under which they will be grown
Control of photoperiod is also important to prevent dormancy or to control plant development

19. Soil and containers Requirement
A uniform medium that adequately supports the plants, has suitable pH, well buffered and sufficiently porous
Inhibitors or dramatic shift in pH in medium can adversely affect root growth
Larger container is better
Peat plugs or small foam blocks are recommended

20. Diseases Very essential
Plantlet is generally suitable to diseases- causing organisms
High humidity is conducive to the growth of many plant diseases causing fungi and bacteria
An integrated approach of sanitation and application of pesticides is generally used:
Disinfested medium, new or disinfested container and benches
New poly-ethylene covers
Clean hand
Clean and disinfested instruments

21. Temperature
The temperature of the air and growing medium are generally controlled
Adjusting the amount of shading and humidity can aid in temperature control
Ventilation and fan system
Fog and air condition
The temperature of the root zone is important to encourage root growth

22. Nutrient
Nutrient can be originally from the Media, if the media consists of soil, sand and compost
Fertilizer may be incorporated or top dressed in slow release form
A soluble complete fertilizer diluted to ¼ to ½ the recommended rate is recommended

23. Direct field acclimatization
It is possible in some species
Vanilla, Teak, Potato
A covering of 40 mesh screen
Only 6 – 14% survived
Survival and yield varied among clones

24. Rooting and acclimatization
In vitro rooting
Ex vitro rooting
In vitro rooting
Disadvantage:
Lack root hair
Died and collapsed after plantlet was removed from culture, however new lateral and adventitious root formed during acclimatization
The transition zone between root and shoot was abnormal
The vascular connection were poorly form
Restricted water uptake
Labor intensive and expensive

25. Direct rooting during acclimatization
Ex-vitro rooting
Direct rooting during acclimatization
Attention must be paid to humidity, light and temperature
Treatment with root inducing growth regulators may be required prior to acclimatization
No agar adheres to the base of the cutting

26. Post Culture Behavior Dwarfs Color changes or mosaic pattern (Chimera)
Growth habit changes
Change in productivity
Cause:
When shoots are derived from dedifferentiated cell
Rapid proliferation of single cells or multi-cellular primordia through organogenesis or embryogenesis
In vitro process or by added biochemical and stress agent
Temporary or heritable deformities

27. Cause Variation that existed in the source plant Genetic changes
Chimera
Non chimeric chromosomal variation
Genetic changes
Mitotic abnormality
Somatic crossing over
polyploidy
Epigenetic or physiological effect
A non heritable change in phenotype that occurs in a substantial percentage of the propagated population through an inducible directed and reversible process

28. Plant with two or more distinct genotypes
Chimera
Plant or plant part composed of genetically different sort of cells as a result of mutation or grafting
Plant with two or more distinct genotypes

29. Chimera Cell origin Mimicked by variegated or mosaic forms
Mericlinal chimera
a section of one or two of the histogenic layers are different
Sectorial chimera
all histogens in a sector are different
Periclinal chimera
one histogen is different from the others
Mimicked by variegated or mosaic forms
Due to en-even distribution of viruses in plant tissue

30. Histogen
Cell layers in all higher plant tissue that trace back to distinct layers in the apical meristem
3 layers in angiosperm
LI an outer epidermal layer
LII an internal tunica layer
LIII a cortical layer
LII layer produce gametic tissue and some surrounding maternal tissue
The remaining maternal tissue is also formed by LIII and LI
Root derived from LII and LIII layers

32. GROWING POINT (APICAL MERISTEM)
Layer
Gives rise to:
L-I
Epidermis of all organs;  Monocot leaves - L-I  contributes to the outermost region of the leaf mesophyll giving rise to a strip along the leaf margin.  Dicot leaves - L-I usually gives rise to only the colorless epidermis, thus cannot be seen; sometimes L-I gives rise to small islands of tissue along the margin
L-II
Stem and roots:
Outer and inner cortex and some of vascular cylinder leaves: mesophyll in outer region of leaf
L-III
inner cortex, vascular cylinder and pith  leaves: mesophyll in central region of leaf 

33. Non chimeric chromosomal variation
Breakage in heterochromatic region
Somatic crossing over (mitotic exchange between homologous chromosomes)
Gene amplification due to mutagenic agent

34. Permanent genetic change
Somaclonal variation
Genetic change
Polyploidy  Aneuploidy and breakage
Micronucleus formation  Bi- or multi nucleate cells
Duplication  Recombination
Inversion  Amplification
Simple base pair change  Organelle genome variation
Isozyme differences
Expression of cryptic transposable element
Change in chromosome structure

35. Definition
Euploidy
An even increase in number of genomes (entire chromosome sets)
Aneuploidy
An increase in number of chromosomes within a genome

36. Euploid Euploid Symbol Somatic (2n) monoploid x (ABC) diploid 2x
(ABC)(ABC)
triploid 3x
(ABC)(ABC)(ABC)
autotetraploid 4x
(ABC)(ABC)(ABC)(ABC)
allotetraploid
2x+2x'
(ABC)(ABC)(DEF)(DEF)

37. Aneuploid Aneuploids Symbol Somatic (2n) Description nullisomic 2x-2
(AB)(AB)
(missing a chromosome set)
monosomic
2x-1
(ABC)(AB)
(missing a chromosome)
double monosomic
2x-1-1
(AB)(AC)
(missing 2 different chromosomes)
trisomic
2x+1
(ABC)(ABC)(A)
(additional chromosome)
double trisomic
2x+1+1
(ABC)(ABC)(A)(B)
(2 additional different chromosomes)
tetrasomic
2x+2
(ABC)(ABC)(A)(A)
(2 additional chromosomes - same)
trisomic-monosomic
2x+1-1
(ABC)(AB)(A)
(missing a chromosome + additional chromosome)

38. Plant variation from dedifferentiated cell
Mitotic asynchrony caused by growth regulator effect on DNA biochemistry (2,4,5-T; 2,4-D; antibiotic; alkaloid; physical mutagen)
Disorientation or dysfunction of the mitotic apparatus (spindle fiber)
Selection pressure due to the change in plant’s environment

39. 2,4-D Increase growth and reduced cell cycle time
Stimulate DNA synthesis
Endo-reduplication lead to nuclear fragmentation
Increased mitotic crossing over
Increase poly-ploid

40. Temporary alterations
Altered flowering, sex expression, fertility and yield
Increased vigor and root-ability
Increased branching
Expression of off-type and off-color phenotypes
Alter susceptibility to diseases and biochemical including herbicide
Rejuvenation

41. Rejuvenation Bring back to youthful appearance (juvenile) Juvenility:
The condition of a seedling plant that prevents flowering or sexual gameto-genesis