Trouble shooting

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Trouble shooting

• Contamination
• Ecophysiology
• Hyperhydricity
• Browning
• Variability

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Contamination

• Virus
• Bacteria
• Fungi
• Mycoplasma
• Micro arthropods and thrips

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Diseases due to plant viruses cause enormous losses in both agriculture and horticulture. In tissue culture it is especially important to work with 'virus free' material, as micro-propagation from virally infected explants could lead to millions of diseased plants. However, the term 'virus free' is abused. Virus free does not exist and only virus testedplants can be produced, after indexing for one specific virus. A way to eliminate viral infection in tissue culture is to use meristem culture. In order to eliminate viral infection it is necessary to know which viruses plant materialis infected with. Viruses are divided into 35 families or groups. Over 700 viruses are known.
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We do not have the possibility to prove a culture
is free of bacteria. Sometimes symptoms of
bacterial infestation are visible on the plant
material or on/in the culture medium. Often
visible symptoms appear late in culture or after
a few subcultures. They may also not appear at
all, but create problems in the early stages of
acclimatization. Not all bacteria are
pathogenic, they may even have beneficial
effects.
Agrobacterium Bacillus Enterobacter
Pseudomonas Lactobacillus Staphylococcus
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Major sources of fungal infestations are non adequate surface sterilization contaminations during transfer manipulations introduction of fungal spores by mites and/or thrips. Fungal contamination is rather easily detected with naked eye.
Detection of Mycoplasma is problematic because they are difficult in culture, so that indexing is not easy.
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In most laboratories infestation by micro arthropodes and/or thrips has been observed. It is one of the major problems. In se those organisms are not that harmfull, but they cause problems due to the introduction of fungal spores and bacteria in the cultures.
fungal contamination by mites
mites
thrips
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Life cycle Female mites attach themselves to their host and suck their food. From that moment on the body of the mite starts to grow and eggs are produced, which develop to embryos and mites. Ten days later the first mites are born. The first new born mite is always a male, followed by about 150 females. The latter are directly fecundated by the male. This explains the exponential growth of a mite colony.

mites one vessel can containthousands of mites a swollen female,filled with eggs a burst female, filled with offspring
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Ecophysiology
Water retention Water transport Gases
Headspace composition Determining factors
Effects
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Measuring the water loss of leaves as a function of time gives a good idea about the water retention capacity of the plants. The figure illustrates that,compared to greenhouse grown plants, tissue culture plants have a poorly developed water retention capacity. Bydecreasing the RH in the container to 75 the water loss curve follows a pattern closer to that of the green houseplants. This improved water control is due to better functioning stomata.
Water retention capacity
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Important water fluxes are present in a tissue culture container. The sugar concentration on which the shoots are maintained has no major effect on the water fluxes, while the agar concentration has. The higher the agar concentration the more water is translocated via the shoots to the surroundings. These shoots have better water retention.
(A) experimental setup In a meli jar a small cup is fixed in the centre 90 ml of stage 2 medium is poured around the cup, 7 ml of stage 2 medium prepared with 3H2O is pored in the cup, and evaporation from the cup is prevented. (B) water fluxes, over a 48-h period, in a tissue culture container with stage 2 of Rosa multiflora. The values (in ) represent the redistribution of the 3H2O-activity, initially added to the small cup. Data are averages of different experiments using different agar and sucrose concentrations and plants of different age in culture.
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component greenhouse environment container atmosphere
O2 22 as low as 4
N2 77 up to 87
CO2 365 - 1000 ppm up to 20
water vapour 60-85 100
ethylene 5 ppb - 100 ppb gt 2 ppm
The headspace compostion in a tissue culture container can be significantly different from the greenhouse atmosphere, as shown in the table. Other volatiles are present in smaller amounts.
The headspace composition influences plant growth and development e.g. dwarf growth or albinism hyperhydricity necrosis autotrophy/mixotrophy ethylene damage Factors determining the gaseous compositonof an in vitro culture container
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Factors determing the gaseous composition of an
in vitro culture container
1. Plant species, cultivar and stage of development day/night pattern photosynthetic capability stress sensitivity 3.Culture-container characteristics of body and closure device material, structure, thickness, shape, etc. permeability for gases and water vapour mechanisms of gas transfer headspace volume and plant density
2. Medium formulation state liquid or solidified partition and diffusion coefficients (solubility) external carbon source, hormonal balance, nutrient supply use of inhibitors of biosynthesis and action, air scrubbers 4. Environmental conditions temperature, pressure, light, relative humidity and gaseous composition in- and outside the container (spatial variation) day- and night cycles influence of bottom cooling, ventilation, etc. Contamination
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Tissue culture containers are closed as tight as possible toprevent contamination. However, in a completely closed container accumulation of gases can occur, whichpossibly lead to abnormalities in plant cultures such as growth reduction (Fig. 1), albinism (Fig. 2) or necrosis. Fig. 3 and 4 illustrate that large variations in time and withindifferent containers occur in the levels measured for thedifferent gases, probably dueto differences in the container (type, closure and gas exchange). Fig. 1 growth reduction Fig. 2 Albinism

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Hyperhydricity is a physiological disorder
occuring in plant tissue cultures. It occurs to a
lower or higher extent. When it occurs to a lower
extent no major problems are encountered up to
the weaning stage. When it occurs to a higher
extent serious problems can be faced in the
different stages.
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Symptoms of hyperhydricity can be minimised by
Medium ingredients media solidified with a higher concentration of a gelling agent, as well as the use of a gelling agent with a higher gel strength lowering the cytokinin concentration
Container characteristics a not too tight closure device as well as gas-permeable membranes allow increased exchange of water vapor and other gasses with the surrounding environment, and thus alter the headspace of the container
Environmental conditions bottom cooling increasing light intensity cold storage on a medium without BA
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Explants and or the medium frequently turn brown or black after isolation. Growth is inhibited and the tissue usually dies.
Growth inhibition is most severe in species that naturally contain high levels of tannins or other hydroxyphenols.
Browning may be influenced by the time of year at which the tissue is explanted, the presence of contaminants, the sterilisation procedure, etc..
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Tissue blackening occurs through the action of
copper containing oxidase enzymes (e.g.
polyphenoloxidases and tyrosinases) which are
released or synthesised and presented with
oxidative conditions when tissues are wounded.
Substrates for these enzymes vary in different
tissue. Enzymes and substrates are normally
retained within different compartments and come
together when cells are injured or moribund.
Phenols have an important natural function in
regulating IAA oxidation (auxin protector). The
toxicity of phenols is probably due mainly to
reversible hydrogen bonding to proteins.
Irreparable growth inhibition occurs when
phenols are oxidised to highly active quinone
compounds which then cyclise, polymerise and/or
oxidise proteins to form increasingly melanic
compounds.
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Blackening of tissue and the medium can oftenbe
prevented by one of several different approaches
removing the phenolic compounds produced
modifying the redox potential inactivating
phenolase enzymes reducing phenolase activity
and substrate availability.
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Somaclonal Variation

• Somaclonal variation is a general phenomenon of
  all plant regeneration systems that involve a
  callus phase
• There are two general types
• Heritable, genetic changes (alter the DNA)
• Stable, but non-heritable changes (alter gene
  expression, epigenetic)
• With or without mutagen

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Somoclonal variation in Rhododendron plants derived from adventitious shoots The phenomenon of high variability in individuals from plant cell cultures or adventitious shoots has been named somaclonal variation. Somaclonal variation is not restricted to, but is particularly common in callus-derived regenerants. The variations can be genotypic or phenotypic, which in the later case can be either genetic or epigenetic in origin. Typical genetic alterations are changes in chromosome numbers (polyploidy and aneuploidy), chromosome structure (translocations, deletions and duplications) and DNA sequence (base mutations). Typical epigenetic related events are gene amplification and gene methylation. If no visual, morphogenic changes are appearant, other plant screening procedures must be applied. There are both benefits and disadvantages to somaclonal variation
Benefits The major likely benefit of somaclonal variation is in plant improvement. Somaclonal variation leads to the creation of additional genetic variability. Characteristics for which somaclonal mutants can be enriched during in vitro culture include resistance to disease pathotoxins, herbicides and tolerance to environmental or chemical stress, as well as for increased production of secondary metabolites.
Disadvantages A serious disadvantage of somaclonal variation occurs in operations which require clonal uniformity, as in the horticulture and forestry industries where tissue culture is employed for rapid propagation of elite genotypes.
Ways of reducing somaclonal variation Different steps can be taken to avoid somaclonal variation. It is well known that increasing numbers of subculture increases the likelihood of somaclonal variation, so the number of subcultures in micropropagation protocols should be kept to a minimum. Regular reinitiation of clones from new explants might reduce variability over time. Another way of reducing somaclonal variation is to avoid 2,4-D in the culture medium, as this hormone is known to introduce variation.
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Somaclonal/Mutation Breeding

• Advantages
• Screen very high populations (cell based)
• Can apply selection to single cells
• Disadvantages
• Many mutations are non-heritable
• Requires dominant mutation (or double recessive
  mutation) most mutations are recessive
• Can avoid this constraint by not applying
  selection pressure in culture, but you lose the
  advantage of high through-put screening have to
  grow out all regenerated plants, produce seed,
  and evaluate the M2