Problems in tissue culture Culture contamination Vitrification Acclimatization

Problems in tissue culture ü Culture contamination ü Vitrification ü Acclimatization ü Post culture behavior

Culture Contamination ü Two sources: 1. Carry over of microorganism on the surface or in tissue of explants 2. Through faulty procedures in the laboratory

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

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

Endophytic microorganisms ü Intercellular endophytic microorganism Virus Viroid Fastidius prokaryotes ü 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

Aspect of quality control ü Awareness of the range and natural history of possible contaminant of the crop, including specific pathogen ü Adequate preparation of the donor plant including treatment to reduce or eliminate pathogens ü 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 ü 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 ü An awareness that the spectrum of contaminating microorganism may alter with time in culture ü Monitoring of progeny based on sampling of production

Detection and identification methods Test Non specific tests: Application 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

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

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

Factors associated to vitrification Physical and chemical state of the medium 1. Type of culture media 2. Gelling agents 3. Organic component 4. Inorganic component 5. Growth regulators 6. Relative humidity 7. Environmental condition 8. Various addition

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

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

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

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

Important aspect in the control environment acclimatization üHumidity üLight üSoil and container üDiseases üTemperature üNutrient

Control humidity ü 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 ü Fogging ü The use of a humidifier ü Placing plantlets in an enclosed area that will water vapor ü The use of anti-transpirants to reduce water losses

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

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

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

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

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

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

Rooting and acclimatization 1. In vitro rooting 2. 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

Ex-vitro rooting Direct rooting during acclimatization üAttention must be paid to humidity, light and temperature üTreatment with root inducing growth regulators may be required prior to acclimatization üNo agar adheres to the base of the cutting

Post Culture Behavior ü ü 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

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

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

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

Histogen q Cell layers in all higher plant tissue that trace back to distinct layers in the apical meristem q 3 layers in angiosperm ü LI an outer epidermal layer ü LII an internal tunica layer ü LIII a cortical layer q LII layer produce gametic tissue and some surrounding maternal tissue q The remaining maternal tissue is also formed by LIII and LI q 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 üBreakage in heterochromatic region üSomatic crossing over (mitotic exchange between homologous chromosomes) üGene amplification due to mutagenic agent

Permanent genetic change ü Somaclonal variation ü 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 ü Change 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

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

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

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

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

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

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