Practical Considerations in the Development of a Botanic Garden Micropropagation Laboratory

Michael F. Fay

Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3AB, U.K.

Abstract

Micropropagation and other in vitro methods are now widely recognised as being useful for the propagation of plants in botanic gardens. These techniques were originally developed with agricultural and horticultural crop species, but have now also been used with a wide range of wild species, including orchids, ferns, woody plants, succulents and carnivorous plants. In addition to micropropagation in its strict sense, other techniques available include in vitro seed germination, regeneration of plants from callus cultures, dual culture with symbiotic fungi and micrografting. The use of these techniques has allowed the propagation of many species which prove problematic with conventional horticultural methodology. The requirements for setting up micropropagation laboratories are discussed, including consideration of growth room, general laboratory and glasshouse facilities. The advantages and disadvantages of the different techniques and the types of plants for which they are appropriate are also considered.

Introduction

The effective conservation of plant germplasm requires the interaction of various technologies. Falk (1990) used the term 'integrated strategies', and discussed the use of reserves, land restoration, botanic gardens, seed banks etc. Which strategies are the most appropriate will vary from species to species.

Mistretta et al. (1991) discussed the role of botanic gardens in the conservation of plant genetic resources, with reference to the role of taxonomy, horticulture, seed banking and education. Since their inception, botanic gardens have been centres for the development of propagation techniques, and can therefore provide information necessary for propagation of a wide range of species. This accumulated knowledge can be useful in conservation programmes which include restoration, introduction or reintroduction of plants by propagating large numbers, either from seeds, or by vegetative increase in the number of ramets.

During the last thirty years, micropropagation and other in vitro techniques have become more widely used in commercial horticulture and agriculture for the mass propagation of crop plants and for conservation of genetic resources, particularly with those crops which are vegetatively propagated or have recalcitrant seeds which cannot be stored under conventional seed bank conditions (George & Sherrington, 1984; Dodds, 1991, George, 1993). Likewise, in vitro culture is being used in an increasing number of botanic gardens for the propagation and conservation of wild plant species (Fay, 1992a).

Orchids have been grown from seed using in vitro methods at Kew since the early 1960s, and in 1974, a micropropagation laboratory was established to carry on this work and to widen the range of plant groups being cultured. Currently, approximately 500 species are being propagated in vitro at any one time, with representatives from many different families. The techniques used include seed germination, micropropagation, meristem culture and callus culture. In vitro culture has proved particularly useful with groups of plants which are difficult to propagate using conventional techniques (Fay 1994).

In vitro culture can have major advantages over conventional propagation techniques in the management of some of these species. Rapid multiplication under controlled, pathogen-free conditions can be achieved by the inclusion of plant growth regulators or hormones. In this way, large numbers of shoots can be produced from small quantities of initial material, in some cases as little as one bud or seed.

Because aseptic conditions are used, problems with the international movement of plant material are considerably reduced, and most countries will accept batches of plants in vitro with a phytosanitary certificate, without requiring a rigorous quarantine period. In this way, plants reach their final destination more quickly and hopefully in better condition than they would if shipped as cuttings or rooted plants. Plants listed on CITES (Convention on International Trade in Endangered Species) Appendices which have been propagated in vitro generally require less documentation than other material. By distributing this material, survival in cultivation is more likely to be ensured.

In this paper, the role of in vitro methods in botanic gardens and ideas about how to set up a micropropagation unit are discussed.

In vitro methods

When working with rare and endangered species, the amount of available plant material can be very small, and this can place restrictions on the choice of methods. Seeds are preferred to vegetative material as the source of propagation material as a wider genetic base can thus be maintained. However, in some species, seed is not readily available and therefore vegetative material has to be used.

Surface sterilisation

As in vitro cultures are normally axenic (i.e. they are pure cultures of one organism), it is necessary to eliminate possible contaminants from the surface of the explants. Such contaminants include fungal, bacterial and algal spores. Various sterilants are used to achieve this, the most commonly used compound being sodium hypochlorite (NaOCl), either as a chemical-grade reagent or as a commercial household bleach formulation. Other sterilants used include calcium hypochlorite, mercuric chloride and hydrogen peroxide. A wetting agent such as Tween 80 is often included to improve contact between the sterilant and the plant tissue. Concentration of, and time of exposure to, the sterilant vary considerably, depending on the nature of the explant. In general, seeds can be subjected to more stringent conditions than most vegetative material. (George 1993). Orchid seed capsules that have not split can be flamed in alcohol and then cut open to release the seeds that are normally sterile. In this way, damage to the seeds by excessive bleaching can be avoided.

Choice of method

Depending on the number of seeds available, either one plant is produced from each seed or a proliferation stage is included, giving rise to a clone of plants from each seed.

In the case of vegetative material, tissue with existing meristematic activity is used where possible, normally apical or lateral buds with surrounding tissues. Culture on media containing cytokinins causes the dormant buds to start growing and to produce shoots. In this way, many shoots can be produced from the original bud. When the required number of shoots has been produced, shoots are transferred to rooting media, either without growth regulators or with auxins. Meristem culture provides a useful variation on this type of culture where plants are infected with viruses, as in many cases, the meristem itself does not become infected with virus. Combination with high temperature treatments/antiviral compounds can improve the efficacy of meristem culture (George 1993).

Where tissue with existing meristematic activity is not available, it is possible in some cases to induce adventitious or de novo shoot formation (organogenesis) or somatic embryogenesis, either directly on an explant e.g. leaf tissue, or via a callus stage. (Somatic embryogenesis is the formation of structures that resemble zygotic embryos de novo from somatic cell cultures). However, when working with plants of conservation importance, it is generally considered wise to avoid callus culture as this can result in somaclonal variation i.e. genetic variation in plants derived from somatic tissue (Fay 1994). With endangered plants, it is usual to try and maintain genetic integrity in tissue culture. In very reduced populations which have reached a genetic "bottle-neck", somaclonal variation has been suggested as a method of generating new vigour (Bramwell 1990; Jacobsen & Dohmen 1990), but there is as yet no published literature showing that somaclonal variation has had a beneficial effect on endangered species.

Media

There are many different media used for plant tissue culture, and these are extensively reviewed in George et al. (1987a, b), but it is possible to make some general comments about their components.

Major minerals (macronutrients)

Nitrogen, phosphorus, potassium, calcium, magnesium and sulphur have long been known to be indispensable for plant growth, and chlorine and sodium are normally included, although their role is less clear (George & Sherrington 1984). Nearly all media contain salts of these elements, but the salts used vary between formulations. The form of N appears to be more critical than for most other elements, and many authors have discussed the relative benefits of nitrate and ammonium N. Many woody plants, for example, grow far better on media with lower nitrate concentrations than that found in standard media, such as Murashige and Skoog's MS medium (1962). In many early media formulations, N was only supplied as nitrate. However, Knudson (1922) showed that orchid seeds could be germinated on media containing ammonium ions, and Morel (1960) used Knudson's C medium, containing both ammonium and nitrate N to obtain the first recorded protocorm-like body proliferation. For a consideration of organic N, see below in the section on organic additives.

Minor minerals (micronutrients)

This group of elements are essential for the normal physiological functioning of plants. For example, iron, manganese, zinc, molybdenum and copper are essential for chlorophyll synthesis and chloroplast function, boron is involved in meristematic activity, and cobalt is the metal component of Vitamin B12. Iron is often used in a chelated form to keep it in solution. Other elements used in media include aluminium, nickel and iodine. Effects of micronutrients on plant tissue cultures are discussed in George & Sherrington (1984).

Energy sources

Until recently, nearly all plant tissue cultures were grown on media containing a sugar as an energy source, as very few cell and tissue lines had been shown to be fully autotrophic in vitro, i.e. they are not capable of satisfying their own carbohydrate requirements by photosynthesis. Sucrose is the most frequently used sugar, but glucose and other sugars are also used.

Recent work has shown that many cultures have the ability to become autotrophic under suitable conditions, but photosynthesis is restricted by suboptimal CO2 levels and by sucrose in the medium. This can be overcome by increasing the CO2 level in culture vessels, and by reducing the sucrose concentration, or omitting it altogether.

Where technology for controlling the gaseous environment is available, this technique allows the production of plants that more normally and are easier to wean to glasshouse conditions (Kozai 1991). At present, however, the necessary equipment is too expensive to be used in most micropropagation laboratories.

Other defined organic additives

Most media contain small quantities of other organic compounds, including vitamins, myo-inositol and amino acids. In general, intact plants synthesise these compounds themselves, but they are beneficial or necessary in at least some tissue cultures (George & Sherrington 1984). Myo-inositol and thiamine (vitamin B1) are normally considered to be the two most important compounds in this group.

Support systems

Solidified media are often used for plant tissue cultures. The most frequent method of producing such media involves the inclusion of agar, an extract from certain seaweeds (e.g. Gelidium spp.). As the purity of agar varies and grades contain significant quantities of nutrients, including carbohydrates, amino acids and mineral salts, it is advisable to use the same brand of agar routinely, otherwise variable results may be obtained. In scientific experiments looking at the role of specific compounds, it has been suggested that other support systems should be used as these other compounds could influence the results (George & Sherrington 1984). Other purified polysaccharides are sometimes used including Gelrite, which produces an agar-like gel in the presence of certain divalent cations (e.g. Mg2+ or Ca2+). Non-gelatinous substrates have been used to provide a 'platform' for plant tissues, including filter paper bridges, cellulose plugs, rockwool and glass beads (Pierik 1987).

Other components

These five classes of components are included in nearly all plant culture media, but there are also several other classes of compounds that are included in some media. The most important group of these are the plant growth regulators that are used to control the growth pattern of the cultures. These and others are discussed below.

Growth regulators

Three main groups of growth regulators are used in media. These are cytokinins, auxins and gibberellins. Each group has naturally occurring and synthetic members, the natural ones often being known as hormones. George & Sherrington (1984) give an extensive account of the use of these compounds in tissue cultures.

Cytokinins are normally used for their ability to induce either shoot proliferation, by breaking dormancy in lateral buds (suppressing apical dominance) or adventitious meristem/shoot formation.

Auxins have two major roles in plant tissue culture. Firstly, as in conventional horticulture, they can be used as rooting compounds. This can be useful for plants which do not readily root on artificial growth media. Secondly, they can be used to induce callus formation from which plants can, in many cases, be regenerated. This regeneration can either be via organogenesis (adventitious shoot formation) or via somatic embryogenesis. Somatic embryogenesis is normally induced by the stronger synthetic auxins such as 2,4-dichlorophenoxyacetic acid or picloram.

Manipulation of the auxin:cytokinin ratio can be used to alter the growth and developmental pattern of tissue cultures, and thus obtain the desired effect.

Gibberellins are only normally used in vegetative propagation where shoot elongation following proliferation proves difficult to achieve. In addition, they can be used to improve rates of seed germination, both in vitro and ex vitro.

Non-defined organic additives

Many media used with orchids, and sometimes with other types of plants, contain components of which the exact chemical composition is unknown and variable. These include yeast extract, coconut milk, banana pulp, potato extract or pineapple juice. Products such as hydrolysed casein (mixtures of amino acids and short-chain peptides) are purer, but still of variable composition. From a scientific viewpoint, the addition of such components to the medium is rather unsatisfactory, as it precludes the possibility of investigating the effects of individual components of the medium with any degree of accuracy. However, they have been shown to promote plant growth in vitro (e.g. Arditti 1982; Butcher & Marlow 1989). In addition to their nutrient role, some of these components also appear to have growth regulatory properties e.g. coconut milk has cytokinin-type activity (George & Sherrington 1984).

Activated charcoal and antioxidants

These types of additives are used particularly in cases where the explants have a tendency to excrete large quantities of waste products into the medium. As a result of the tissues being cultured in a sealed vessel, it is impossible for these to leach away, and so they accumulate and can become toxic to the plant. A wide range of substances are involved, but they have been referred to generically as 'phenolics' and they often cause a darkening/browning of the medium around the cut end of an explant. This is predominantly a problem with woody plants.

Why activated charcoal limits the extent of this problem is not fully understood. It is possibly a beneficial effect of light reduction, the absorption of inhibitory substances or a combination of both these effects. It has been shown to have a beneficial effect on the growth of a wide range of plants and the germination of some seeds (George 1993).

The inclusion of charcoal in media, as for some other components described above, makes the composition of the medium undefined due to its variable composition and the variable way in which it absorbs other compounds from the medium. The latter can be the cause of major differences in aliquots from the same batch of medium which have been stored under different conditions or for different lengths of time. This makes accurate replication of results more difficult to achieve. (Ebert & Taylor 1990).

A different solution to the problem of exudates is to try and prevent their occurrence, rather than absorbing them when they have been produced. To try to achieve this, anti-oxidants such as ascorbic acid and citric acid can be added to the medium (George 1993).

Equipment

When setting up a tissue culture or micropropagation laboratory, it is possible to consider doing this at different levels (high tech vs. low tech). Clearly, this will depend on the funding available and the projects involved. Units already in existence vary from 'state of the art' research laboratories to ones with very basic facilities. The major utilities and pieces of equipment to be found in tissue culture laboratories are discussed below.

A source of pure water is necessary for preparation of media, sterilants and for washing plant material etc. This can be distilled or ultrapure water or water produced by reverse osmosis. In areas where the mains water is low in minerals and relatively pure, then deionised water is also an option.

A chemical balance for weighing out media components is required, and should be accurate to 3 decimal places. As some of the chemicals used in this type of laboratory can be hazardous to health, precautions should be taken to avoid the exposure of personnel to such risks. In many countries this area is covered by national legislation, and therefore it is not possible to make recommendations covering this subject in such an article. However, where possible, suitable fume hoods should be used.

A pH meter is necessary for adjusting the pH of media, sterilants etc. It should be accurate to 1 decimal place. If large amounts of media are to be produced, then an autoclave is necessary for sterilising media and also pure water, instruments etc. If the laboratory is on a much smaller scale, then a pressure cooker can be used instead. Where large quantities of media are made and need to be stored, this should be in a cold room (c. 4ºC) in the dark. For smaller quantities of media and stock solutions etc. a domestic refrigerator can be used. Some media components (e.g. some vitamins and antibiotics) need to be stored frozen (-20ºCº).

Microwave ovens are useful for dissolving agar in media prior to autoclaving. Magnetic stirrers prove useful for mixing media and for improving surface sterilisation of plant material, by facilitating contact between the sterilant and the tissue.

After surface sterilisation, all activities should be carried out under sterile conditions. This is normally achieved by the use of laminar airflow benches, which filter the air, removing bacterial and fungal spores, providing a clean airflow in which to work. In this way, vessels can be opened and plant material manipulated without risk of contamination.

Binocular microscopes are used in processes such as dissecting out shoot meristems, removing seed coats etc.

The types of vessels used varies greatly between laboratories. At Kew, the main types used are honey jars (two sizes), test tubes, Petri dishes and some other disposable vessels. A range of standard glassware, including beakers, flasks, pipettes, measuring cylinders etc., is also required.

In the design of the laboratory, it is important to make sure that there are sufficient power points. This is particularly so in the light of the increase in the use of computers in recent years (see below under the heading 'Computing'). This increase in the number of machines has necessitated the provision of a number of extra power points in the laboratory at Kew.

Growthroom facilities

The environmental conditions required will depend on the species being grown and on the ambient climatic conditions. Those used in the main growth room at Kew are 22°C and a long day (16h light/8h dark). The light intensity ranges from 400 to 4000 lux in different parts of the room. Under these conditions, we find that it is possible to grow a wide range of plants from tropical and temperate parts of the world. Lower temperatures are not good for the tropical species and higher temperatures are not good for the temperate species. Light quality can also affect plant growth, and therefore consideration should be given to this. However, where a wide range of plants is being grown in the same room, it is not possible to optimise this or other conditions for each species, and so we use a mixture of different types of fluorescent tubes, providing a wide range of different wavelengths.

In addition to the growth room, we have several incubators, which we use for vernalising plants or providing other variations in environmental conditions.

Greenhouse facilities

Weaning (or acclimatising) plants back to conventional conditions from in vitro culture can prove problematic with some plants, due to problems of dehydration and infection with pathogens. Greenhouse facilities should take this into account, and provision of high levels of humidity in either a misting/fogging system and the use of free draining composts and fungicides can alleviate these problems. These aspects are discussed in more detail in a recent article (Fay 1992b). As for growthrooms, the exact set-up will depend on the local climatic conditions.

Long-term conservation

In vitro culture allows for the long-term maintenance of plant germplasm under disease-free conditions, and this can be achieved in different ways:

- Continuous growth under "normal" growthroom conditions;

- Reduced growth by alteration of the culture conditions;

- Suppression of growth at very low temperatures (cryopreservation).

Sections on each of these subjects are included in Dodds (1991). Other useful reviews have been written by Villalobos et al. (1991), Wilkins et al. (1990) and Withers et al. (1992).

Networks of Units

Gradually more and more botanic gardens are establishing micropropagation units, many with the aim of propagating rare and endangered species. Some, in areas of high genetic diversity, have the remit of propagating plants endemic to their own country or region. Others, in areas of low biodiversity, like Kew, can propagate plants from other places as well.

We are keen to foster an increased awareness of what is being done in these units, and this is facilitated by the production of Botanic Gardens Micropropagation News - a newsletter containing articles from different units around the world. Eight issues have been produced (as of April 1995). Anyone who would like to receive subsequent issues should write to the author.

Computing

Increasingly, computers are becoming part of everyday life in botanic gardens. In addition to their use for word processing and graphics, we are in the process of producing databases of literature references and of micropropagation protocols developed at Kew. The second database is still in the developmental stage, but it is hoped that this will allow the dissemination of such information more easily.

References

Preface  |  Contents List  |  Congress Report  |  Workshop Conclusions  |  List of Authors