Animal and plant cell cultureKultura animalnih i biljnih stanica

plant part/biljni dio

Izv.prof.dr.sc. Nataša Bauer Andreja Škiljaica Mateja Jagić

nbauer@biol.pmf.hr

Plant tissue culture is a collection of

techniques used to maintain or grow

plant cells, tissues or organs under

sterile conditions (in vitro) on a nutrient

culture medium of known composition.

Basic to all plant biotechnologies and

an exciting area of basic and applied sciences

Definition

Plant tissue culture relies on the totipotency, the characteristic of many plant cells to have the ability to regenerate a whole plant.

Single cells, plant cells without cell walls (protoplasts), pieces of leaves, stems or roots can be used to generate a new plant on culture media given the required nutrients and plant hormones.

The production of exact copies of plants that produce desirable traits (particularly good flowers, fruits, or grow well under stress conditions).

The production of multiple plants in the absence of seeds. The regeneration of whole plants from plant cells that have been genetically modified. The production of plants in sterile containers that allows them to be moved with

greatly reduced chances of transmitting diseases, pests, and pathogens. The production of plants from seeds that otherwise have very low germination To clear particular plants of viral and other infections To quickly multiply desired plants as 'cleaned stock' for horticulture and agriculture.

Crop improvement Haploid and Triploid production, In Vitro Fertilization, Hybrid Embryo Rescue, Variant Selection

Clonal propagation Micropropagation

Virus elimination Shoot Tip (Meristem) Culture

Germplasm conservationProduction of phytochemicals Regeneration of plants from fused plant cells

Somatic Hybridization Cybridization

Regeneration of plants from genetically manipulated cells - GMO

Plant tissue culture technics

See presentation on youtube:https://www.youtube.com/watch?v=6y13hYGPi8Q

Application in Fundamental ResearchPlant tissue culture implies all conducted studies under strictly controlled conditions and manipulation of clons (often). Studies at the gene level – gene eliminations, downregulation or overexpression

- gene function research, - genetic engineering, - mutagenesis and selection of cell lines with specific properties

Studies of the role of various agents on plant growth and developmentCell-level- effects on genetics and epigenetics and - effects on differentiation of somatic cellsSecondary metabolism- effect on synthesis and regulation of metabolismDevelopmental biology- mechanism of action of hormones (mutants, tumors),- mechanisms of differentiation and morphogenesis,- synthesis of cell wall materials (protoplast culture),- properties of plasma membrane- transport of substances across membrane

Short history of plant tissue culture

1902. Haberlandt made the first attempts to culture differentiated cells with the purpose to achieve divisions of these cells and obtain complete plants, to verify the concept of totipotency.

- did not achieved his goals, but for the first time, he gave

postulates and principles of plant tissue culture

*First plants regenerated from single

cell achieved 1965. Gottlieb Haberlandt

1854-1945

1922. Kotte (pea) and Robbins (maize) suggest the meristematic cells from shoot and root tip as good starting material for plant tissue culture

Kotte Robbins

In 1934. White announced the establishment of continuously growing root cultures of tomato.

First defined medium – White medium

After auxin discovery Gautheret obtained continuously growing callus cultures (in 1939)

Gautheret

• Continuously growing culture implies SUBCULTURING of plant materialGrowth of tissue on medium is limited while the nutrients are exhausted and the medium dried. Cells and tissues need to be subcultured on fresh medium. Theoretically, the plant cell/tissue culture can be infinite if it is regularly subcultured.

Murashige & Skoog

• Murashige & Skoog demonstrated chemical regulation of organogenesis – by adding different concentrations of auxin and cytokinin to medium they directed root or shoot regeneration in tobacco.

• Formulated the most extensively used plant tissue culture medium popularly called MS medium (in 1962.)

Strong improvement of plant tissue culture after MS medium application

Totipotency - the potential to regenerate the entire plant from one plant cell

• During growth and development, the cells differentiate and stop to divide. Many plant cells are not terminally differentiated, could be dedifferentiated induced to divide and redifferntiate!

• Thanks to this ability, the tissue and / or complete plant can be regenerated from cotyledons, hypocotyl, leaves, roots, stamens…

Differentiation, dedifferentiation and redifferentiation

• The cells derived from apical meristem (root, shoot or cambium) mature to perform specific functions. This act leading to maturation is termed differentiation.

• The living differentiated cells can regain the capacity to divide mitotically under certain conditions. The sum of events, that confer capacity to divide are termed dedifferentiation. Dedifferentiation is an important biological phenomenon whereby cells regress from a specialized function to a simpler (basic or meristematic).

Major postulates of plant tissue culture

• A dedifferentiated cell/tissue can act as embryonic/meristematic cells and could regenerateinto new type of cells/tissue. This phenomena is called redifferentiation.

Amazing regeneration ability of plants

Laser ablated SAM

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Laser ablated RAM

Front Plant Sci. 2014; 5: 142.

Plasticity – ability of plants to adapt to different environmental conditions by changing their metabolic activity, growth and development

Plants have a remarkable ability to alter their development in response to myriad environmental cues or stress. This phenotypic plasticity allows them to continually adapt to their local environment, a necessity for plants as sessile organisms.

Plasticity gives plants the ability to optimize growth in varied environments

Tissue Culture of Sequoia

Major postulates of plant tissue culture

Major requirements of plant tissue culture

Selection of suitable explant

Addition of necessary nutrients and removal of undesirable metabolic products from medium

Aseptic materials and manipulation

Highly controlled environmental conditions

Explant: most plants, at most stages of the life cycle have some populations of cells that are totipotent

Any plant part could be used as explant in establishment of plant tissue culture

Explants may contain different types and variable concentrations of metabolites, growth regulators, transcription factors etc. important for establishment of plant cell culture.

Culture media

Medium is source of:

Water

MineralsMacroelementsMicroelements

Organic compoundsVitaminsGrowth regulatorsAmino acids(Occasionally: complex mixtures of

undefined composition)

The growth, development and morphogen response of an explant in culture depends on its genetic/epigenetic properties, environment and culture medium composition

Success of culture largely depends on the selection of right culture medium

The culture medium is defined by the type and amount of inorganic components

In practice we usually prepare macronutrient stock solutions (each compound separately) that are 100 X concentrated, and a unique stock solution for micronutrients that is 1000 times concentrated

Macroelements –required in millimolar quantities (mM)

Nitrate (NO3-) – critical for protein biosynthesis

Ammonium (NH4+) reduced nitrogen form

Organic nitrogen

- 25-60 mM

Potassium (K+) – main cellular ion, necessary for cell division, protein and chlorophyll biosynthesis

- 20-30 mM

Phosphorous (PO43-) – important for enzyme activation/inactivation and synthesis of nucleic acids

- 1-3 mM

Calcium (Ca2+) – constituent of cell wall, involved in regulation of hormone response and enzyme activity

- 1-3 mM

Magnesium (Mg2+) – component of chlorophyll – important for photosynthesis, enzymes cofactor

- 1-3 mM

Sulphur (SO42-) – important protein component

- 1-3 mM

Microelements, essential but required in small quantities

Fe2+ 1 mM – part of enzymes, respiratory electron carrier through cytochrome and peroxidases and catalase

Mn2+ 5-30 mM – important for chloroplast membrane structures and enzyme activity

Zn2+ – important for chlorophyll synthesis, enzymes

cofactor

BO33—important for sugar movement in plants

Cu 2+ 0.1 mM – important for oxidative enzymes: part of cytochrome oxidase, tyrosinase and ascorbate oxidase

MoO4 - 1 mM – essential for nitrogen metabolism (component of nitrate reductase)

Co 2+ 0.1 mM – necessary for nitrogen fixation

Na2EDTA

I-

Ni

Al

Si

Organic compounds

To achieve the best growth the medium need to be supplemented with different

organic compounds.

Carbohydrates

Most cultures are unable to photosynthesize because of absence of chlorophyll, poorly

developed chloroplasts, limited CO2 and suboptimal light intensity. Addition of sugars

significantly promote plant tissue growth and is essential for cultivation of non-photosynthetic

tissues.

Different sugar types could have specialized role in somatic embryogenesis, androgenizes, etc.

-Sucrose - the main transport form of sugar in plants. When autoclaved, hydrolysis into

glucose and fructose.

-Glucose, fructose, maltose etc.

Addition of 20 - 50 g/L in media is recommended since higher concentrations suppress

growth due to high osmolarity

Myo-inositol – important in signal transduction, second messenger, important in

auxin action and storage, crucial in cell wall formation, role in uptake and ion utilization

Amino acids

- Gly and Gln are most widely used

Vitamins

-Thiamine (vitamin B1) – most widely used. Coenzyme, important for the synthesis

of some amino acids and for Krebs cycle.

-Nicotinic acid (niacin or B3) – important for photosynthesis

-Pyridoxine (B6)

-Ascorbic acid (vitamin C) – antioxidants

Organic compounds

Activated charcoal

https://doi.org/10.1016/j.biotechadv.2008.08.003

Often used in tissue culture to improve cell growth and

development

• Critical role in micropropagation, orchid seed germination,

somatic embryogenesis, anther culture, synthetic seed

production, protoplast culture, rooting, stem elongation,

etc.

• Promotary effects on morphogenesis, mainly due to its

irreversible adsorption of inhibitory compounds in the

culture medium and substancially decreasing the toxic

metabolites, phenolic exudation and brown exudate

accumulation.

• Involved in a number of stimulatory and inhibitory activities

including the release of substances naturally present in

activated charcoal which promote growth, adsorption of

vitamins, metal ions and plant growth regulators, including

abscisic acid and gaseous ethylene.

• Necessary to trigger various types of growth and differentiation.• Explant may have certain levels of endogenous hormones, but to evoke certain

response exogenous application of growth regulators is essential. • The nature and quantity of growth regulators is varied according to the variety of plant,

nature of explant, stage of culture.• In order to develop a tissue culture protocol for a new species, various types and

concentrations of growth regulators in several permutations and combinations need to be tested.

Plant growth regulators

Hormones - organic substances that are synthesized in the plant and in low concentrations stimulate, inhibit, or in any way quantitatively alter growth, usually at a non-synthesis site.

The term growth regulator implies hormones and synthetic components that act like natural hormones

Main plant growth regulators:Auxins - Involved in stem elongation, tropism, apical dominance, abscission

and rooting.- In cell culture are used root initiation and in combination with cytokinins for shoot proliferation(IAA, NAA, IBA);- In higher concentration for callus and somatic embryogenesis induction (2,4-D)

Cytokinins- Important for cell divisions, modification of apical dominance and shoot differentiation- In culture used to trigger cell division, differentiation od adventiousshoots from callus and organs and for multiple shoot induction- There are two types of cytokinins: adenine-type cytokinins and phenylurea-type cytokinins- Most commonly used are BAP, kinetin, 2iP and zeatin

Plant growth regulators

Synthesized in shoot apices, leaf primordia

and developing see from the amino acid

tryptophan

Most adenine-type cytokinins are

synthesized in roots

Gibberellins- Less commonly used, GA3 is most often used- Stimulate internode eleongation, brake dormancy and induce meristem growth

Abscisic acid- Naturaly occuring growth inhibitor required for normal growth, development and embrio maturation- Widly used in stress tolerance tests

Ethylene- Producen by aging or in stress- Influence organogenesis and embriogenesisHormones are used in comcentration from 0.001 – 10 µMIn most cases thay have to be desolved in ethanol. NaOH or DMSO and than diluted

vith water to appropriate concentration

Plant growth regulators

Balance of the growth regulator

Au

xin

Cyt

oki

nin

EmbryogenesisInduction of roots on cuttings

Adventive roots from callusCallus initiation

Induction of axillary shoots

The effect of cytokinin on the

induction of shoots on leaf explantThe effect of auxin on root

induction on stem and leaf explant

The response is dependent on the age

and location of the explorationDifferential response of different plants

Hormone / growth regulator activity is not always the same:The same chemical can cause a different response on different plants.

Some parts of the same plant react differently to the same chemical

Liquid and solidified culture media

In static liquid cultures the tissue will get submerged and die od anaerobic conditions. For solidification of medium different substances could be used.

Gelling agents:Agar - Red Algae Polysaccharide (0.6 - 1%)

- different purity in the market - significantly influences the outcome of the experiment- Solidification depend on pH and salt concentration- dissolves at 100 °C and solidified at 45-50 °C

Gelrite or Phytagel or Gellan gum- Polysaccharide from Pseudomonas, cleaner than agar- Uses 0.2-0.35%, dissolves at 100 °C, and solidified at 55 °C

AgaroseMixtures (Phytagar)Different mechanical materials are used to support the explant - bridges from filter

paper, sand, wool, glass wool ...

pH of the medium

Greatly influence the uptake of ingredients, solubility of salts and gelling efficiency

Usually 5.0-5.8

- It has to be measured and ajusted when all the substrate components are added, except agar because agar can clog the pH electrode

The agar will not solidified the medium if e pH is below 5 Gelrite is used at low pH as it solidifies but higher concentrations are needed

During autoclaving and growth pH gradually decreases. If the pH has to be maintained constant the buffering agent may be added, eg MES

Media preparation

Critical step in tissue culture Weight powdered components (sugar, inositol, protein hydrolysates, agar ...) Add distilled water to the vessel (less than the final medium volume) and dissolve the

powdered components, except the agar! Add macroelements and microelements, each from the in advance prepared stock solutions Add vitamins, amino acids, growth regulators, and other additives from the stock solutions Measure and adjust the pH The final volume is made up with distilled water Add the agar (depending on the selected dish, melt the agar in the microwave and divide) Autoclave the medium at 121 ° C, 10 min

OPTION:Add a thermolabile component to the cooled medium after autoclavation (at 60 ° C)Divide the substrate (under sterile conditions) into suitable sterile containers and leave it to solidified.

Sterile medium after autoclaving or subsequently melted in a microwave oven

Add a thermolabile, filter sterilized component, in a cooled medium, under sterile conditions

Pouring the medium in sterile containers, under sterile conditions.

Preparation of medium with thermolabile component

Storage of prepared culture media

Smaller volumes (up to 100 mL) are stored at room temperature for up to 2 weeks or in a fridge for up to 6 weeks.

Larger medium volumes (500 mL and more) are stored at room temperature for up to 6 weeks or in a fridge for up to 3 months.

Media with thermolabile components are always stored in the fridge for a shorter time (up to 4 weeks).

The best way to add thermolabile components is to add them into the nutrient medium immediately before use. In this case, the sterile nutrient medium is dissolved in a microwave oven or water bath and a cooled (60 ° C), and thermolabile component is added.

The agar solidified media can be repeatedly dissolved, while those with Gelrite cannot.