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Plant tissue culture, the growth of plant cells outside an intact plant, is a technique essential in many areas of the plant sciences. Cultures of individual or groups of plant cells, and whole organs, contribute to understanding both fundamental and applied science.
It relies on maintaining plant cells in aseptic conditions on a suitable nutrient medium. The culture can be sustained as a mass of undifferentiated cells for an extended period of time, or regenerated into whole plants.
Designing a strategy to culture cells from a plant for the first time can still seem like a matter of trial and error, and luck. However, the commercial production of valuable horticulture crops by micropropagation, which relies on tissue culture, shows that it exists in the routine, as well as experimental, world.
In the School of Biological Sciences at the University of Liverpool, we have experience over many years with the techniques and applications of plant cell culture.
What is Plant Tissue Culture?
Plant cells can be grown in isolation from intact plants in tissue culture systems. The cells have the characteristics of callus cells, rather than other plant cell types. These are the cells that appear on cut surfaces when a plant is wounded and which gradually cover and seal the damaged area.
Pieces of plant tissue will slowly divide and grow into a colourless mass of cells if they are kept in special conditions. These are:
initiated from the most appropriate plant tissue for the particular plant variety
presence of a high concentration of auxin and cytokinin growth regulators in the growth media
a growth medium containing organic and inorganic compounds to sustain the cells
aseptic conditions during culture to exclude competition from microorganisms
The plant cells can grow on a solid surface as friable, pale-brown lumps (called callus), or as individual or small clusters of cells in a liquid medium called a suspension culture. These cells can be maintained indefinitely provided they are sub-cultured regularly into fresh growth medium.
Tissue culture cells generally lack the distinctive features of most plant cells. They have a small vacuole, lack chloroplasts and photosynthetic pathways and the structural or chemical features that distinguish so many cell types within the intact plant are absent. They are most similar to the undifferentiated cells found in meristematic regions which become fated to develop into each cell type as the plant grows. Tissue cultured cells can also be induced to re-differentiate into whole plants by alterations to the growth media.
Plant tissue cultures can be initiated from almost any part of a plant. The physiological state of the plant does have an influence on its response to attempts to initiate tissue culture. The parent plant must be healthy and free from obvious signs of disease or decay. The source, termed explant, may be dictated by the reason for carrying out the tissue culture. Younger tissue contains a higher proportion of actively dividing cells and is more responsive to a callus initiation programme. The plants themselves must be actively growing, and not about to enter a period of dormancy.
The exact conditions required to initiate and sustain plant cells in culture, or to regenerate intact plants from cultured cells, are different for each plant species. Each variety of a species will often have a particular set of cultural requirements. Despite all the knowledge that has been obtained about plant tissue culture during the twentieth century, these conditions have to be identified for each variety through experimentation.
Utilising anther culture to select for cold hardiness
Crosses between distantly related species can bring together novel gene combinations. However, the hybrid offspring can be few in number, genetically unstable and require years of further selection and screening before any advantageous characteristics can be brought near to commercial use.
Anther culture (androgenesis), to generate haploid plants from pollen microspores, is one way to shorten this process. It allows novel allele combinations, particularly ones involving recessive characters, to be assessed in intact plants. Useful individuals can then be developed into homozygous and fertile plants through chromosome doubling techniques, and brought into a breeding programme.
We have recently been involved in a collaborative project with the Institute of Grassland and Environmental Research (IGER) to use this approach to improve cold-tolerance and fodder quality in grazing grasses. Crosses between Lolium multiflorum ( Italian ryegrass) andFestuca arundinacea (tall fescue) should offer valuable combinations of characteristics. TheLolium species should provide good growth characteristics, while the Festuca provides stress-tolerance. One hybrid individual (Festulolium) resulting from such a cross had already shown drought-tolerance characteristics. However, the out-breeding nature of these grass species, along with the hexaploid genome of F. arundinacea and autotetraploid L. multiflorumindicated that a lengthy breeding programme might be necessary.
The research project therefore aimed to produce androgenic plants from the existing pentaploidFestulolium plant and assess them for cold tolerance. This exploited the expertise in tissue culture at the University of Liverpool together with experience in breeding for stress-tolerance at IGER.
Anthers containing immature pollen (microspores) are the starting material for androgenesis. Flowers have to be selected at the correct developmental stage, which varies from species to species. In addition, some individual genotypes may not be amenable to anther culture, or require specific pretreatments. Careful microscopy and testing of successful pre-treatments of related species are therefore necessary when dealing with a new species. For the Graminae, microspores must be at the mononucleate stage and no pre-treatment is necessary.
The cut flowers were surface sterilised and opened in sterile conditions under a binocular microscope. The anthers were dissected and transferred to a solid nutrient medium. Large numbers could be placed on each petri dish. Callus developed, which was transferred to a different medium to initiate embryos and so generate haploid plants.
Over 200 androgenic plants were produced at Liverpool, each originating from a different microspore. Each therefore represented a genetically different individual. Testing for phosphoglucosisomerase, where a different isozyme was contributed by each of the five chromosome groups within the Festulolium plant, indicated that the pollen-derived plants had a wide variety of chromosome combinations from each of the parents of the hybrid. The freezing-tolerance of these plants varied considerably, with three individuals able to survive the extreme cold of -14 degrees Celsius. When the chromosome complement of two of these plants was examined using genomic in situ hybridisation (GISH), they carried virtually the whole genome of F. pratensis, a parent of F. arundinacea noted for its freezing-tolerance.
Unfortunately, the fertility of these two plants was not restored by chromosome doubling, so that they could not be used for further breeding. However, they demonstrated the potential of androgenesis for rapid assessment of the genetic potential available from a difficult breeding combination, indicating that this type of wide cross revealed characters of cold and drought tolerance that were worth pursuing
CASE STUDY 2
Using somaclonal variation to select for disease resistance
lant tissue cultures isolated from even a single cell can show variation after repeated subculture. Distinct lines can be selected with their own particular morphology and physiology. It suggests that the tissue culture contains a population of genotypes whose proportion can be altered by imposing an appropriate selection pressure. This variation can be transmitted to plants regenerated from the tissue cultures, and is called somaclonal variation. It provides an additional source of novel variation for exploitation by plant breeders.
The carrot cultivar Fancy was used in our laboratory to generate a series of 197 regenerant progeny lines. These plants showed considerable morphological variation. They were tested for resistance to the leaf spot pathogen Alternaria dauci, which can cause total necrosis of mature leaves. They had a greater degree of variation in response than the parental cultivar, including some more resistant lines.
Scanning electron micrographs of surface of carrot leaf 3 days after inoculation with A. dauci. (LHS) germination from multiseptate conidiospore (RHS) penetration of hyphae through epidermal surface rather than through stomata. Scale bar = 10 micrometres.
One symptom of the disease is loss of chlorophyll and total soluble polyphenol compounds. These reduce to a low level 6 days after inoculation of excised leaves with A. dauci spores when compared with uninoculated leaves. Regenerant progeny with high chlorophyll levels maintained higher chlorophyll levels after challenge with A. dauci. After self-pollinating selected high- and low-chlorophyll regenerant plants, this characteristic was inherited by their progeny, suggesting that the capacity to resist this infection is inherited.
CASE STUDY 3
Demonstration of tissue culture for teaching
Regenerated African violets (Saintpaulia ionantha)
The starting point for all tissue cultures is plant tissue, called an explant. It can be initiated from any part of a plant - root, stem, petiole, leaf or flower - although the success of any one of these varies between species. It is essential that the surface of the explant is sterilised to remove all microbial contamination. Plant cell division is slow compared to the growth of bacteria and fungi, and even minor contaminants will easily over-grow the plant tissue culture. The explant is then incubated on a sterile nutrient medium to initiate the tissue culture. The composition of the growth medium is designed to both sustain the plant cells, encourage cell division, and control development of either an undifferentiated cell mass, or particular plant organs.
The concentration of the growth regulators in the medium, namely auxin and cytokinin, seems to be the critical factor for determining whether a tissue culture is initiated, and how it subsequently develops. The explant should initially form a callus, from which it is possible to generate multiple embryos and then shoots, forming the basis for plant regeneration and thus the technology of micropropagation. The first stage of tissue culture initiation is vital for information on what combination of media components will give a friable, fast-growing callus, or a green chlorophyllous callus, or embryo, root or shoot formation.
There is at present no way to predict the exact growth medium, and growth protocol, to generate a particular type of callus. These characteristics have to be determined through a carefully designed and observed experiment for each new plant species, and frequently also for each new variety of the species which is taken into tissue culture. The basis of the experiment will be media and protocols that give the desired effect in other plant species, and experience.
The strategy for designing a medium to initiate tissue culture, showing how growth regulators and other factors modulate development, can be demonstrated using the African Violet, a popular house plant. Leaf sections are the source of explants. This demonstration is regularly carried out by a student class, and gives reliable results. Sterile supplies are provided from central facilities, and provision of sterile working areas (for example, in laminar flow hoods) is an advantage, although cultures can be initiated in an open laboratory with careful aseptic technique. The standard precautions used during any laboratory work involving chemicals or microbes should be adopted. If you are in any doubt about safety hazards associated with this demonstration, you should consult your local safety adviser.
Step 1 - selection of the leaves
Leaves are cut from healthy plants, leaving a short length of petiole attached. They should be selected to each yield several explants of leaf squares with approximately 1 cm sides. The youngest and oldest leaves should be avoided.
Wash the dust off the leaves in a beaker of distilled water, holding the leaf stalk with forceps.
Step 2 - surface sterilisation and preparation of the explants
This part of the procedure should be carried out in a sterile working area, or with meticulous aseptic technique.
The leaf, with the petiole still attached, should be immersed in 70% ethanol for 30 seconds, then transferred to a sterile petri dish. Sterile scissors and forceps are then used to cut squares from the leaf as explants, each with approximately 1 cm sides.
The explants are transferred into a 10% hypochlorite bleach solution for 5 minutes, gently agitating once or twice during this time. They are then washed free of bleach by immersing in four successive beakers of sterile distilled water, leaving them for 2-3 minutes in each.
Three explants are placed on each petri dish of growth medium (see table and below), with the upper epidermis pressed gently against the surface of the agar to make good contact.
The petri dishes are sealed with plastic film to prevent moisture loss, and incubated at 25oC in 16h light/8h dark.
Step 3 - assessment of tissue culture development
The explants are incubated for 4 - 6 weeks, and inspected at weekly or fortnightly intervals. The growth of obvious bacterial or fungal colonies indicates contamination, and data from such cultures is obviously suspect. The development of dark brown tissue cultures can also be a consequence of contamination.
The media used in the demonstration are designed to show the effects of auxin, cytokinin, sucrose and mineral salts on development. The media were based on the well-known Murashige and Skoog inorganic medium, with additions as shown in this table.
These pictures show typical results, after about 8 weeks on each medium. To summarise, multiple adventitious buds form on the control medium, leading to many small shoots on the upper surface where the leaf is not in contact with the medium.
Absence of sucrose inhibits this production. Shoot production is also limited on the low sucrose concentration, but comparable with the control at high sucrose.
At zero and low levels of cytokinin, callus forms where the leaf surface is in contact with the medium, while at high levels, shoot formation is stimulated.
At zero and low levels of auxins there is a stimulus to shoot formation, but at high concentrations, large numbers of roots are formed.
At low and zero levels of MS salts, there is no growth at all.
These very obvious variations demonstrate the importance of a carbon and inorganic salt source for plant growth, as well as the effect of the auxin:cytokinin ration on the control of plant development.
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