Problems in tissue culture

Culture contaminationVitrification

AcclimatizationPost culture behavior

Culture Contamination

Two sources:

1. Carry over of microorganism on the surface or in tissue of explants

2. Through faulty procedures in the laboratory

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

Organisms associated with plant surface

Fungi including yeast Bacteria Mollicutes (mycoplasmas, spiroplasmas and

related organism)

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

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

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

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

Vitrification

TranslucencyHyperhydration

SucculencyGlassines

Change into a glassy appearanceProduce fragile plants which have a glassy and hyperhydrous appearance

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

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

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

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

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

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

Important aspect in the control environment

acclimatization Humidity

Light Soil and container

Diseases Temperature

Nutrient

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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 

Non chimeric chromosomal variation

Breakage in heterochromatic region

Somatic crossing over (mitotic exchange between homologous chromosomes)

Gene amplification due to mutagenic agent

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

Definition• Euploidy

An even increase in number of genomes (entire chromosome sets)

• Aneuploidy An increase in number of

chromosomes within a genome

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)

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)

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

2,4-D

Increase growth and reduced cell cycle time

Stimulate DNA synthesisEndo-reduplication lead to nuclear

fragmentationIncreased mitotic crossing overIncrease poly-ploid

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

Rejuvenation

Bring back to youthful appearance (juvenile)

Juvenility:The condition of a seedling plant that prevents flowering or sexual gameto-genesis