Problems in tissue culture
description
Transcript of Problems in tissue culture
![Page 1: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/1.jpg)
Problems in tissue culture
Culture contaminationVitrification
AcclimatizationPost culture behavior
![Page 2: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/2.jpg)
Culture Contamination
Two sources:
1. Carry over of microorganism on the surface or in tissue of explants
2. Through faulty procedures in the laboratory
![Page 3: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/3.jpg)
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
![Page 4: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/4.jpg)
Organisms associated with plant surface
Fungi including yeast Bacteria Mollicutes (mycoplasmas, spiroplasmas and
related organism)
![Page 5: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/5.jpg)
Endophytic microorganisms Intercellular endophytic microorganism
VirusViroidFastidius prokaryotes
Intracellular endophytic microorganismL-forms of common plant associated bacteriaFastidius bacteria associated with plant vascular tissuevector transmitted and may be spread by contact between infected and healthy plantthey are capable of clonal propagation
![Page 6: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/6.jpg)
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
![Page 7: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/7.jpg)
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
![Page 8: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/8.jpg)
Detection and identification methodsTest ApplicationNon specific tests:Culture indexingDNA stainingLeaf dip electron MicroscopeGel electrophoresis
Cultivable bacteriaMycoplasmas and related prokaryotesVirusesViroids
Specific tests:ELISADNA probesRapid diagnostic kitFatty acid profiling
Viruses and bacteriaAll organismsBacteriaBacteria
![Page 9: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/9.jpg)
Vitrification
TranslucencyHyperhydration
SucculencyGlassines
Change into a glassy appearanceProduce fragile plants which have a glassy and hyperhydrous appearance
![Page 10: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/10.jpg)
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
![Page 11: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/11.jpg)
Factors associated to vitrificationPhysical and chemical state of the
medium
1. Type of culture media2. Gelling agents
3. Organic component4. Inorganic component
5. Growth regulators6. Relative humidity
7. Environmental condition8. Various addition
![Page 12: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/12.jpg)
AcclimatizationProcess during which plants or other
organisms become adjusted or accustomed to a new climate or situation as a result of natural processes
Hardening-offMoving the complete plants to
greenhouse or fieldNot unique to micro-propagation
![Page 13: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/13.jpg)
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
![Page 14: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/14.jpg)
Common issueAgar has to be thoroughly washed from
the rootPesticide may be phytotoxic to some
species of micro-propagated plantletGradually reduce the RH or amount of
mistMaintain light level in the greenhouse at
50% shade before plants are transplanted to the field
![Page 15: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/15.jpg)
Acclimatization Control environment acclimatization
Specific place that all environmental conditions can be control either automatically or manually
Direct field acclimatizationTransferring the rooted plantlet directly to the field
![Page 16: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/16.jpg)
Important aspect in the control environment
acclimatization Humidity
Light Soil and container
Diseases Temperature
Nutrient
![Page 17: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/17.jpg)
Control humidityAvoid 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
FoggingThe use of a humidifierPlacing plantlets in an enclosed area that will
water vaporThe use of anti-transpirants to reduce water
losses
![Page 18: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/18.jpg)
LightWhile 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
![Page 19: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/19.jpg)
Soil and containersRequirement
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 betterPeat plugs or small foam blocks are
recommended
![Page 20: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/20.jpg)
DiseasesVery essentialPlantlet is generally suitable to diseases-
causing organismsHigh 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 benchesNew poly-ethylene coversClean handClean and disinfested instruments
![Page 21: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/21.jpg)
TemperatureThe temperature of the air and growing
medium are generally controlledAdjusting the amount of shading and
humidity can aid in temperature controlVentilation and fan systemFog and air conditionThe temperature of the root zone is
important to encourage root growth
![Page 22: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/22.jpg)
NutrientNutrient 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 formA soluble complete fertilizer diluted to ¼ to ½ the
recommended rate is recommended
![Page 23: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/23.jpg)
Direct field acclimatization It is possible in some speciesVanilla, Teak, Potato
A covering of 40 mesh screenOnly 6 – 14% survived
Survival and yield varied among clones
![Page 24: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/24.jpg)
Rooting and acclimatization
Disadvantage:Lack root hairDied and collapsed after plantlet was removed from culture, however new lateral and adventitious root formed during acclimatizationThe transition zone between root and shoot was abnormalThe vascular connection were poorly formRestricted water uptakeLabor intensive and expensive
1. In vitro rooting2. Ex vitro rooting
In vitro rooting
![Page 25: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/25.jpg)
Ex-vitro rooting
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
Direct rooting during acclimatization
![Page 26: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/26.jpg)
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
![Page 27: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/27.jpg)
CauseVariation that existed in the source
plant Chimera Non chimeric chromosomal variationGenetic changes Mitotic abnormality Somatic crossing over polyploidyEpigenetic 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
![Page 28: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/28.jpg)
ChimeraPlant or plant part composed of
genetically different sort of cells as a result of mutation or grafting Plant with two or more distinct
genotypes
![Page 29: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/29.jpg)
ChimeraCell origin
Mericlinal chimeraa section of one or two of the histogenic layers are differentSectorial chimera
all histogens in a sector are differentPericlinal chimera
one histogen is different from the othersMimicked by variegated or mosaic formsDue to en-even distribution of viruses in
plant tissue
![Page 30: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/30.jpg)
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
![Page 31: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/31.jpg)
![Page 32: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/32.jpg)
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 Stem and roots: inner cortex, vascular cylinder and pith leaves: mesophyll in central region of leaf
![Page 33: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/33.jpg)
Non chimeric chromosomal variation
Breakage in heterochromatic region
Somatic crossing over (mitotic exchange between homologous chromosomes)
Gene amplification due to mutagenic agent
![Page 34: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/34.jpg)
Permanent genetic change Somaclonal variationGenetic 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 elementChange in chromosome structure
![Page 35: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/35.jpg)
Definition• Euploidy
An even increase in number of genomes (entire chromosome sets)
• Aneuploidy An increase in number of
chromosomes within a genome
![Page 36: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/36.jpg)
EuploidEuploid 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)
![Page 37: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/37.jpg)
AneuploidAneuploids 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)
![Page 38: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/38.jpg)
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
![Page 39: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/39.jpg)
2,4-D
Increase growth and reduced cell cycle time
Stimulate DNA synthesisEndo-reduplication lead to nuclear
fragmentationIncreased mitotic crossing overIncrease poly-ploid
![Page 40: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/40.jpg)
Temporary alterationsAltered flowering, sex expression, fertility and
yield Increased vigor and root-ability Increased branchingExpression of off-type and off-color phenotypesAlter susceptibility to diseases and
biochemical including herbicideRejuvenation
![Page 41: Problems in tissue culture](https://reader035.fdocuments.in/reader035/viewer/2022062315/5681610c550346895dd05c88/html5/thumbnails/41.jpg)
Rejuvenation
Bring back to youthful appearance (juvenile)
Juvenility:The condition of a seedling plant that prevents flowering or sexual gameto-genesis