The influence of physical factors on growth and development

Growth conditions

Most important environmental conditions in a tissue culture growth room are:

Light Temperature Humidity Oxygen

THE LIGHT REQUIREMENTS OF PLANTS

Photosynthesis Photomorphogenesis Phototropism

Photosynthesis, photomorphogenesis and photo-tropism are all facilitated by pigments in the tissues, which absorb radiation of particular wavelengths.

LightPAR is a more applicable measurement than other types such as lux because it measures the light spectrum that plant can use for photosynthesis.The amount of light needed for tissue culture explants is much lower compared to plants in grown in the field.Generally, in tissue culture growth rooms, 60-100 mol.m-2s-1 PAR is used.High levels of light will damage the explants.Photosynthesis does not usually take place in tissue culture plants unless you use photoautotrophic plant regions. In cases where there is photosynthesis, it is very limited mainly due to the physiology and anatomy of explants.

Light requirements of plantsThe growth and development of plants is dependent on light for:

1. Photosynthesis2. The process whereby light energy is

converted to chemical energy in the biosynthesis of chemicals from carbon dioxide and water. Photomorphogenesis•The light-induced development of structure or form. •It does not necessarily involve the absorption of light energy, it uses receptor which act as switches to set in motion the morphogenetic processes of plants.

Light

For plants, it is measured in PAR (photosynthetically active radiation) - the number of photons per square meter per second for the spectral range from 400-700 nm.

LUMINOUS FLUX In photometry, luminous flux or luminous power is the

measure of the apparent power of light. It differs from radiant flux, the measure of the total power of electromagnetic radiation (including infrared, ultraviolet, and visible light), in that luminous flux is adjusted to reflect the varying sensitivity of the human eye to different wavelengths of light.

Photosynthetically active radiation

Photosynthetic Photon Flux Density (PPFD):

Photosynthesis photon flux density

Photosynthetically active radiation, often abbreviated PAR, designates the spectral range (wave band) of solar radiation from 400 to 700 nanometers that photosynthetic organisms are able to use in the process of photosynthesis. This spectral region corresponds more or less with the range of light visible to the human eye.

Light PAR is normally quantified as µmol

photons m-2s-1, which is a measure of the photosynthetic photon flux (area) density, or PPFD. PAR can also be expressed in energy units (irradiance, W/m2); this is relevant in energy-balance considerations for photosynthetic organisms. Because photosynthesis is a quantum process, PPFD is generally used by plant biologists.

THE INFLUENCE OF LIGHT ON TISSUE CULTURES

The three aspects of the photo-environment which most clearly influence in vitro growth and morphogenesis, are:

wavelength flux density the duration of light exposure or photoperiod.

Wavelength

Plants absorb blue and red lights, which have the greatest effect on plant growth. Red light:

Photosynthesis Seed germination, seedling growth Flowering, fruit development

Blue light: Photosynthesis Vegetative growth (leaf) growth

Wavelength

Growth rooms are mostly equipped with cool white fluorescent tubes.

Gro-lux tubes are specifically made to plants, but are more expensive.

Traditional radiant light bulbs are not appropriate, since most of the light emitted is heat.

Light Limited reports on the effect of light on explant

growth is available due to the complex relationship between light and plant.

The numerous factors that will affect plant response to light are: Plant species Type of explant (leaf, stem, root, etc) Type of development of the explant (embryo, callus,

meristem, etc.)

Often conflicting results are reported with different plants exposed to similar light conditions.

Blue light

Blue light (420nm) at a lower intensity stimulates callus and shoot development in tobacco explants.

While at higher intensities, it inhibits callus growth and cell division.

The intensity of blue light has the biggest effect on stimulation and inhibition of callus growth.

Red light

In general, red light (660 nm) promotes adventitious shoot formation in most plants.

In addition, red light stimulates adventitious root growth in sunflowers and tobacco more than blue light.

While red light inhibits organogenesis in tobacco.

Light requirements of plantsThe growth and development of plants is dependent on light for:

1. Photomorphogenesis•The light-induced development of structure or form. •It does not necessarily involve the absorption of light energy, it uses receptor which act as switches to set in motion the morphogenetic processes of plants.

Figure 1. Photomorphogenesis as a morphological and as a cellular process. The left photos show the change in form of an Arabidopsis thaliana seedling grown in darkness (top) or in white light. The right hand illustration shows the change in chloroplast structure and diagrams the progress of light signals through two receptor systems, cryptochrome and phytochrome. Adapted from Biochemistry and Molecular Biology of Plants, (c) American Society of Plant Biologists, with permission

Plants Response to Light• Photomorphogenesis

– nondirectional, light-mediated changes in plant growth and development • red light changes the shape of phytochrome and can trigger

photomorphogenesis • Stems go from etiolated (in dark or Pfr) to unetiolated (in light with Pr).

• Photoperiodism– Regulates when seeds of lettue and some weeds. Presence of Pr

inhibits germination, while its conversion to Pfr in red light induces germination

Red light ===> germination Far-red light ===> no germination 

Those seeds not buried deep in the ground get exposed to red light, and this signals germination. 

– Regulates when plants flower; either in the Spring or later in the Summer and Fall.

How Phytochrome Works

Photoperiod Photoperiod is the length of time a plant receives

light in 24 hours.

Photoperiod influences plants in 2 ways:1. Growth of plant is proportional to the length

of time that they are exposed to light.High light (summer) = more growthLow light (winter) = less growth

2. Plants are able to sense changes in the photoperiod and respond accordingly. In nature, photoperiod affects flowering and morphogenesis.

Photoperiod

Photoperiod of tissue culture growth rooms is dependent on the type of explant cultured.

When uncertain, the photoperiod of plants in nature are used in vitro.

Most explants grow well with 14-16 hours of light.

For specific purposes, complete darkness is used (eg., seed germination).

Temperature

In vivo photosynthesis: Increases with temperature up to a point. Although photosynthesis is low in tissue cultured explants,

optimum temperature is still required for growth.

In vivo respiration: Rapidly increases with temperature Sugar, starch and oxygen is converted to CO2 and energy.

Temperature The temperature of a growth room is usually kept constant at 24-28C. Sometimes in experiments, depending on the origins of the explants,

lower temperature (18C) for bulbous species, or higher temperature (28C) for tropical species is chosen.

The temperature within the test tubes is 3-4C higher than the growth room due to irradiance.

Naturally temperatures vary from day to night. Not the same required for tissue culture although this

variation may have different effects on tissue culture (reducing temp in the night may reduce fuel consumption)

Average culture temp 25C (from 17-32)

Temperature Sometimes alternating temperature conditions

may be needed.

This is particularly evident in seed germination.

Common alternating temperature regimes include a 26C daytime temperature, and 15C night temperature.

For example, callus tissue of carrots grows best under a day temperature of 26C and night temperature of 20C.

1. Growth rooms temp usually uniform (all species grown at the same temp). But many species have different growth optimal temp

3. Increase or Decrease of temp

2. Green house effect and in culture vessels

MorphogenesisShoot bud formation:Organogenesis optimumNarrow range b/w speciesInduction at:Low temp eg: begoniaHigh temp eg: pinusNormal temp eg: rapeseed

Pretreatment: Morphogenesis at same temp

as mother plants or Cultures.

Induction at high temperatures

In some plants the temperature for the best induction of adventitious organs is somewhat higher than that required for growth.

In Pinus radiata for example, adventitious buds formed most readily on juvenile explants when they were cultured in a 28°C (day), 24°C (night) regime, whereas the shoots, which were formed grew best at 24/20°C.

By mutagenesis or artificial selection by low temperature induction for saving the cost of heat

Tuber:10 times more tuberAt 20 C than 28 C

Bulbous species:Culture at some tempCauses cessationAnd requiresCold treatment

Cold sensitivity dormancy occurs occasionally in vitro culture or immediately after plantlets are transferred to external environment specially in bulbous species

Dormant plants or bulbils can often be induced to resume growth in vitro or in externa; environment if treated for a few weeks at low temp. (1-10C appropriate)

Induction at normal temperatures

In other plants, shoot regeneration seems to be most effectively induced at temperatures closer to those used for normal tissue culture.

On Brassica napus (colza) flower stalk segments, where adventitious shoots were readily formed at 24°C, low temperatures (13°C for 16 h day, 6°C at night) inhibited subsequent morphogenesis, especially when applied during the first 1-2 weeks of culture.

Selection for a low temperature response.Heating costs involved in the

greenhouse culture of ornamentals make it desirable to obtain varieties with an ability to grow and flower in reduced temperatures.

Direct adventitious root formation The formation of adventitious roots on shoots

is temperature dependent. For example no callus or roots formed on

young shoot tips of Asparagus at 0°, 10° or 15°C, but 20 per cent of cuttings rooted at 20°C, and 45 per cent at 25°C.

In many plants, root induction on the shoot micro-cuttings produced in vitro seems to require a slightly lower temperature than is necessary for shoot multiplication and growth.

LOW TEMPERATURE TREATMENTS

Callus: The proportion of Arabidopsis callus

cultures forming shoots and roots was increased when 9 month old callus was kept for 3-6 days at 4°C before transfer to regeneration medium at the normal culture temperature of 25°C. The weight of roots per callus was also increased by this treatment.

Cold treatments to reverse dormancy

Dormant plants or bulbils can often be induced to resume growth or to germinate, either in vitro or in an external environment, if they are kept for a few weeks at a low temperature (1-10°C has been found to be appropriate).

The induction of flowering

Cold treatments promote the flowering of certain plants in vivo, particularly biennials. The work of Gertsson (1988) has suggested that in vitro culture at cool temperatures may have a similar effect. Plants of Senecio hybridus obtained from shoot cultures maintained at 10°C, flowered earlier, had a greater foliage height, and taller inflorescences, than those from cultures grown at 21°C.

Humidity

Little is known about the influence of growth room humidity and in vitro growth.

However, a growth room with high humidity increases the chances of contamination.

Humidity is usually very high in the test tubes (90-100%).

This causes stomatal malfunction and hyperhydricity.

Relative humidity (R.H.)

Relative humidity (R.H.) is a measure of the amount of water vapour contained in a gaseous atmosphere.

It is expressed as the ratio of the quantity of water an atmosphere actually contains, to that which it could contain when saturated.

THE OPTIMUM HUMIDITY FOR CULTURES

Because cultures grown in high humidities are liable to become hyperhydric, it would be ideal to surround shoot and organ cultures with air that is not fully saturated.

However, a reduction in humidity will increase the risk of desiccation of tissue cultures.

IN VESSELS

The humidity in vessels containing cultured shoots also depends on leaf area development. This is because leaves absorb radiation and commonly exhibit temperatures above the temperature of the air, thereby increasing the driving force for transpiration.

Growth room

Most commonly used conditions:

Temperature: 24ºC - 28ºC

Photoperiod: 16 – 24 hours

Lighting: 60 – 70 μmol m-2 s-1

Oxygen

Oxygen availability is important for in vitro root formation, as is the case in vivo.

For woody plants, it is extremely difficult to regenerate roots when they are in solid medium.

Root formation is much better in a liquid medium.

In this case, the explant is supported on a paper-bridge or on rafts.

Other factors

Certain halophyes might grow better in the presence of high salt or NaCl concentration

Phtotoautotrophic explants might not need carbon source in the medium