The Components of Plant Tissue Culture Media I: Macro- and Micro-Nutrients As an example, in the second edition there was a single chapter on plant growth . PDF | Edwin George's books have become the standard works on in vitro plant propagation. This work will surely maintain that pre-eminence. For this, the third. Request PDF on ResearchGate | Plant Propagation by Tissue Culture: Volume 1. The Background | It is now more than twenty years since the first edition of this.
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Truly encyclopaedic coverage of plant tissue culture and its applications As an example, in the second edition there was a single chapter on plant growth. Search within book. Front Matter. Pages I-XI. PDF · Plant Tissue Culture Procedure - Background Pages PDF · Micropropagation: Uses and Methods. Plant tissue culture or micropropagation technology has made invaluable After many trials and errors in the sixties, plant propagation by tissue culture method.
Plant Propagation by Tissue Culture 3rd Edition. The Background Edited by Edwin F. Catalogue record for this book is available from the Library of Congress. George 2 Micropropagation: Uses and Methods E. George and P. Macro- and Micro-Nutrients 65 E. George and G-J. Thorpe, C. Stasolla, E. Yeung, G-J. Roberts and E. George 5 Plant Growth Regulators I: Introduction; Auxins, their Analogues and Inhibitors I. Machakova, E. Zazimalova and E. Cytokinins, their Analogues and Antagonists J.
Moshkov, G. Novikova, M. Hall and E. George 8 Developmental Biology D. Chriqui 9 Somatic Embryogenesis S. Von Arnold 10 Adventitious Regeneration P.
Gahan and E. Preece 12 Effects of the Physical Environment E.
George and W. Ziv and J. Whilst much of the information in those editions has stood the test of time, inevitably, because of the pace of research, a new edition is clearly timely. This is true, not only because many more species have been the subject of propagation studies, but because the background to the field — with which this volume deals — has changed almost out of all recognition.
In particular, our knowledge of plant development, genetics physiology, biochemistry and molecular biology has expanded exponentially — often through work on mutants of Arabidopsis — and opened up many new avenues for the plant propagator to explore.
Equally, the commercial significance of plant propagation has increased significantly. As an example, in the second edition there was a single chapter on plant growth regulators — in this there are three, reflecting the fact that not only is there more information on those PGRs we recognised in , but that several new ones are now known.
Equally, fifteen years ago we knew little of the molecular basis of plant development e. Because of these factors, it was felt that a different approach was required for this edition. The second edition was researched and written by Edwin George alone but it would now be very difficult for a single author to gain the breadth of expertise necessary to cover all the relevant aspects of this many-faceted subject. Hence, it was decided to adopt a multi-author approach, with chapters written by experts in their fields.
These build upon the sound framework of the previous editions which those with a knowledge of the previous works will recognise. Many sections of the previous work have been retained, but inevitably, apart from up-to-date reference lists, the text has undergone major revision in many areas. Like the previous edition, the current one will appear in two volumes, but coverage has been extended and the order in which subjects are covered has been changed.
Therefore, some topics, previously covered in Part 1, will now be discussed in Part 2.
The ethos of the work is, as before, to produce an encyclopaedic text. The first initiative to begin the new revision of Plant Propagation by Tissue Culture was made by Prof. Cassells and the editors are grateful to him for his early leadership. No work of this size can be accomplished successfully without much goodwill and hard work by the contributors, and to them the editors express their deepest thanks. We also express our sincere thanks to all those who have allowed us to use their material in diagrams and illustrations.
We are very appreciative of the hard work by Dr. Jacco Flipsen of Springer for his support. His areas of interest include Chapter 1. George trained as a botanist at Imperial morphogenesis and micropropagation, mainly of College, London and subsequently gained a PhD, woody plants.
He is an Assistant Professor in the into plant genetic engineering and especially plant Department of Plant Science at the University of tissue culture. His research interests include regulation of growth and functioning of plants experiencing environmental stress; stomatal physiology, root to shoot communication via chemical signalling in plants; environmental physiology of crops and native species; crop improvement for water-scarce environments; irrigation science and enhancing the efficiency of crop water use through novel management techniques.
He has published more than papers in international plant science journals and edited 17 books. Her research interests are in the physiology and morphogenesis of plant organogenesis and somatic embryogenesis in large scale liquid cultures; shoot-malformation, hyperhydricity and the role of oxidative stress in the xi control of plant development in bioreactor cultures for efficient acclimatization and survival ex vitro; bulb and corm development in geophytes cultured in liquid cultures in relation to carbohydrate metabolism.
His interests are in large-scale micropropagation, metabolic pathways and cloning of medicinal plants and plant breeding. Plant tissue culture is the science of growing plant cells, tissues or organs isolated from the mother plant, on artificial media.
It includes techniques and methods used to research into many botanical disciplines and has several practical objectives.
This chapter therefore describes the techniques that have been developed for the isolation and in vitro culture of plant material, and shows where further information can be obtained.
Both organised and unorganised growth are possible in vitro. Unorganised growth is seldom found in nature, but occurs fairly frequently when pieces of whole plants are cultured in vitro. The cell aggregates, which are then formed, typically lack any recognisable structure and contain only a limited number of the many kinds of specialised and differentiated cells found in an intact plant.
A differentiated tissue e. So far, the formation of differentiated cell types can only be controlled to a limited extent in culture. It is not possible, for example, to maintain and multiply a culture composed entirely of epidermal cells. By contrast, unorganised tissues can be increased in volume by subculture and can be maintained on semisolid or liquid media for long periods.
They can often also be used to commence cell suspension cultures. Differentiation is also used botanically to describe the formation of distinct organs through morphogenesis.
It occurs when plant organs such as the growing points of shoots or roots apical meristems , leaf initials, young flower buds or small fruits, are transferred to culture and continue to grow with their structure preserved.
Growth that is coherently organised also occurs when organs are induced. This may occur in vitro either directly upon an organ or upon a piece of tissue placed in culture an explant , or during the culture of previously unorganised tissues. The process of de novo organ formation is called 2. These shoot apices are usually cultured in such a way that each produces multiple shoots.
Each bud is grown to provide a single shoot. The growth of roots, unconnected to shoots: a branched root system may be obtained. It includes the aseptic isolation from whole plants of such definite structures as leaf primordia, immature flowers and fruits, and their growth in vitro.
The shoot apex is typically grown to give one single shoot. George et al.
Embryo culture is quite distinct from somatic embryogenesis see below. These types of cultures are described in more detail later in this chapter. The growth and maintenance of largely unorganised cell masses, which arise from the uncoordinated and disorganised growth of small plant organs, pieces of plant tissue, or previously cultured cells.
Populations of plant cells and small cell clumps, dispersed in an agitated, that is aerated, liquid medium. The culture of plant cells that have been isolated without a cell wall. The culture of complete anthers containing immature pollen microspores.
The objective is usually to obtain haploid plants by the formation of somatic embryos see below directly from the pollen, or sometimes by organogenesis via callus. Pollen cultures are those initiated from pollen that has been removed from anthers. This process is called somatic embryogenesis. To obtain plants by the first two of these methods, it is necessary to treat shoots of an adequate size as miniature cuttings and induce them to produce roots.
The derivation of new plants from cells, which would not normally have taken part in the process of regeneration, shows that living, differentiated plant cells may express totipotency, i.
Totipotency is a special characteristic of cells in young tissues and meristems. It can be exhibited by some differentiated cells, e. Theoretically, plant cells, organs, or plants, can all be cloned, i.
Nevertheless, as will be described in the chapters, which follow, a very large measure of success can be achieved and cultures of various kinds can be used to propagate plants.
Explants Tissue cultures are started from pieces of whole plants. The small organs or pieces of tissue that are used are called explants. Explants can therefore be of many different kinds. The correct choice of explant material can have an important effect on the success of tissue culture. Plants growing in the external environment are invariably contaminated with micro-organisms and pests.
These contaminants are mainly confined to the outer surfaces of the plant, although, some microbes and viruses may be systemic within the tissues Cassells, Because they are started from small explants and must be grown on nutritive media that are also favourable for the growth of microorganisms, plant tissue cultures must usually be established and maintained in aseptic conditions. Most kinds of microbial organism, and in particular bacteria and fungi, compete adversely with plant material growing in vitro.
Therefore, as far as Chapter 1 possible, explants must be free from microbial contaminants when they are first placed on a nutrient medium. This usually involves growing stock plants in ways that will minimise infection, treating the plant material with disinfecting chemicals to kill superficial microbes, and sterilising the tools used for dissection and the vessels and media in which cultures are grown for a review see Cassells and Doyle, Some kinds of plants can, however, be micropropagated in non-sterile environments see Chapter 3.
Isolation and incubation The work of isolating and transferring cultured plant material is usually performed in special rooms or inside hoods or cabinets from which microorganisms can be excluded. Cabinets used for isolation can be placed in a draught-free part of a general laboratory, but are much better situated in a special inoculation or transfer room reserved for the purpose. The accommodation, equipment and methods that are required for successful inoculation and transfer are described in Volume 2.
Cultures, once initiated, are placed in incubators or growth rooms where lighting, temperature and humidity can be controlled. The rate of growth of a culture will depend on the temperature and sometimes the lighting regime adopted. The components of plant tissue culture media are discussed in Chapters 3 and 4.
The compositions of specific media are described in Volume 2. Growth and development of plant cultures usually also depends on the addition of plant growth regulators to the medium see Chapters 5, 6 and 7. Plant growth regulators are compounds, which, at very low concentration, are capable of modifying growth or plant morphogenesis. Many workers define a medium as a completed mixture of nutrients and growth regulators.
This is a rather inflexible method, as growth regulators frequently need to be altered according to the variety of plant, or at different stages of culture, whilst the basic medium can stay unchanged.
It is therefore recommended that nutritional and regulatory components should be listed separately. Plant material can be cultured either in a liquid medium or on a medium that has been partially solidified with a gelling agent see Chapter 4. The method employed will depend on the type of culture and its objective. Solidified media Plant cultures are commenced by placing one or more explants into a pre-sterilised container of sterile nutrient medium.
Some explants may fail to grow, or may die, due to microbial contamination: to ensure the survival of an adequate number, it therefore is usual to initiate several cultures at the same time, each being started from an identical organ or piece of tissue. Explants taken from stock plants at different times of the year may not give reproducible results in tissue culture.
This may be due to variation in the level of external contaminants or because of seasonal changes in endogenous internal growth regulator levels in the stock plant see Chapter Media which have had a gelling agent added to them, so that they have become semi-solid, are widely used for explant establishment; they are also employed for much routine culture of callus or plant organs including micropropagation , and for the long-term maintenance of cultures.
Agar is the most common solidifying agent, but a gellan gum is also widely used Chapter 4. Cultures grown on solid media are kept static.
They require only simple containers of glass or plastic, which occupy little space. Only the lower surface of the explant, organ or tissue is in contact with the medium.
This means that as growth proceeds there may be gradients in nutrients, growth factors and the waste products of metabolism, between the medium and the tissues. Gaseous diffusion into and out of the cells at the base of the organ or tissue may also be restricted by the surrounding medium.
Media 2. Liquid media 2. The cultural environment Plant material will only grow in vitro when provided with specialised media. They are also used in some micropropagation work. Very small organs e. Larger organs such as shoots e. However, some method of support is necessary for small organs or small pieces of tissue, which would otherwise sink below the surface of a static liquid medium, or they will die for lack of aeration.
Systems of support which have been found to be effective and which can be used instead of agarsolidified media are described in Chapters 4. Many tissues and organs, small and large, also grow well unsupported in a liquid medium, providing it is aerated by shaking or moving see below. Some kind of agitation is essential for suspension cultures to prevent cells and cell aggregates settling to the bottom of the flask.
Other purposes served by agitation include: the provision of increased aeration, the reduction of plant polarity, the uniform distribution of nutrients and the dilution of toxic explant exudates Lim-Ho, There are several alternative techniques. Plant cell suspensions can be cultured very satisfactorily when totally immersed in a liquid culture medium, providing it is shaken by a rotary or reciprocating shaking machine or stirred e.
This method may also be used for culturing organs of some plants e. Periodic immersion may be achieved by growing cultured material in tubes or flasks of liquid medium which are rotated slowly.
They were fixed to a wooden wheel, which was rotated so that tissue in the arms of each flask was alternately bathed in medium and drained or exposed to the air Fig.
This technique ensured that callus tissue for which they were used was well aerated. The medium usually became turbid as cells dissociated from the callus and started a cell suspension. Flasks of this sort are seldom used to-day because of their cost. A similar alternating exposure can be achieved by placing calluses in vessels, which are rotated slowly. An alternative to the costly rotating systems to achieve periodic immersion of the cultures, is the increasingly popular temporary immersion system in which static vessels are periodically or temporarily flooded with culture medium Fig.
Medium is pumped from a reservoir container into the culture vessel for experimentally determined time intervals repeated over a 24 hour cycle. This system prevents anoxia and has the advantage that the medium can easily be changed in the reservoir.
Chapter 1 5 Fig. The bioreactor culture is initiated by inoculation with nodes or buds from conventional agar culture. For details of bioreactor design see Fig. Liquid medium in flasks or column bio-reactors fermentors can be circulated and at the same time aerated, by the introduction of sterile air. Shearing forces within air-lift reactors are much less than in mechanically-stirred vessels so that plant cell suspensions suffer less damage.
Bio-reactors are 6 Plant Tissue Culture Procedure - Background used in the pharmaceutical industry to produce high value plant secondary products and to carry out substrate conversions. Low cost bio-reactors developed for micropropagation have been described in detail in Hvoslef-Eide and Preil Fig.
Rather than immersing callus or organ cultures, liquid medium may be slowly dripped onto the growing tissues or applied as a mist and afterwards the liquid drained or pumped away for recirculation Weathers and Giles, A particular advantage of this technique is the ability to grow cultures in a constant and non-depleted medium; nutrients can be varied frequently and rapidly and their availability controlled by altering either concentration or flow rate.
Toxic metabolites, which in a closed container might accumulate and inhibit growth, can be removed continuously.
As complicated apparatus is needed, the method has not been widely used. The relative merits of solid and liquid media and combinations of both are discussed further in Chapter Phenolic oxidation Some plants, particularly tropical species, contain high concentrations of phenolic substances that are oxidised when cells are wounded or senescent.
Isolated tissue then becomes brown or black and fails to grow. The prevention of blackening, which can be a serious problem in tissue culture, is discussed in Chapter Minimum inoculation density Certain essential substances can pass out of plant cells by diffusion. Substances known to be released into the medium by this means include alkaloids, amino acids, enzymes, growth substances and vitamins Street, The loss is of no consequence when there is a large cluster of cells growing in close proximity or where the ratio of plant material to medium is high.
However, when cells are inoculated onto an ordinary growth medium at a low population density, the concentration of essential substances in the cells and in the medium can become inadequate for the survival of the culture. For successful culture initiation, there is thus a minimum size of explant or quantity of separated cells or protoplasts per unit culture volume.
Inoculation density also affects the initial rate of growth in vitro. Large explants generally survive more frequently and grow more rapidly at the outset than very small ones. In practice, minimum inoculation density varies according to the genotype of plant being cultured and the cultural conditions.
For commencing suspension cultures it is commonly about The use of conditioned media can reduce the critical initial cell density by a factor of about 10 Stuart and Street, It is possible to overcome the deficiencies of plant cells at low starting densities by adding small amounts of known chemicals to a medium. For example, Kao and Michayluk have shown that Vicia hajastana cells or protoplasts can be cultured from very small initial inocula or even from individual cells: a standard culture medium was supplemented with growth regulators, several organic acids, additional sugars apart from sucrose and glucose , and in particular, casein hydrolysate casamino acids and coconut milk.
There is often a maximum as well as a minimum plating or inoculation density for plant cells or protoplasts. In a few cases the effective range has been found to be quite narrow. Some effects of inoculation density on morphogenesis are described in Chapter These phases are similarly reproduced by cell suspensions grown in a finite volume of medium a batch culture , where according to a variety of different parameters that can be used to measure growth e. Some differentiation of cells may occur in cell cultures during the period of slowed and stationary 7 growth, but generally it is less marked and less complete than that which occurs in callus cultures.
Cultures cannot be maintained in stationary phase for long periods. Cells begin to die and, as their contents enter the nutrient medium, death of the whole culture accelerates. Somewhat similar patterns of growth also occur in cultures of organised structures. These also cease growth and become moribund as the components of the medium become exhausted.
Subculturing often becomes imperative when the density of cells, tissue or organs becomes excessive; to increase the volume of a culture; or to increase the number of organs e. The period from the initiation of a culture or a subculture to the time of its transfer is sometimes called a passage. The first passage is that in which the original explant or inoculum is introduced. Suspensions regularly subcultured at the end of the period of exponential growth can often be propagated over many passages.
However, many cultures reach a peak of cell aggregation at this time and aggregation often becomes progressively more pronounced in subsequent passages Street, b. Subculture is therefore more conveniently carried out during the stationary phase when cell aggregation is least pronounced. Rapid rates of plant propagation depend on the ability to subculture shoots from proliferating shoot or node cultures, from cultures giving direct shoot regeneration, or callus or suspensions capable of reliable shoot or embryo regeneration.
Thus, even to maintain the culture, all or part of it must be transferred onto fresh medium. Callus subcultures are usually initiated by moving a fragment of the initial callus an inoculum to fresh medium in another vessel. Shoot cultures are subcultured by segmenting individual shoots or shoot clusters. In the early stages of callus growth it may be convenient to transfer the whole piece of tissue to fresh medium, but a more established culture will need to be divided and only small selected portions used as inocula.
Regrowth depends on the transfer of healthy tissues. Decontamination procedures are theoretically no longer necessary during subculturing, although sterile transfer procedures must still be used. However, when using shoot or node cultures for micropropagation, some laboratories do re-sterilise plant material at this stage as a precaution against the spread of contaminants see Volume 2. Cultures which are obviously infected with micro-organisms should not be used for subculturing and should be autoclaved before disposal.
Several kinds of callus may arise from the initial explant, each with different morphogenic potential. Strains of callus tissue capable of giving rise to somatic embryos and others without this capability can, for instance, arise simultaneously from the culture of grass and cereal seed embryos. Careful selection of the correct strain is therefore necessary if cultures capable of producing somatic embryos are ultimately required. Timing of the transfer may also be important, because if left alone for some while, non-embryogenic callus may grow from the original explant at the expense of the competent tissue, which will then be obscured or lost.
Although subculturing can often be continued over many months without adverse effects becoming apparent, cultures of most unorganised cells and of some organised structures can accumulate cells that are genetically changed. This may cause the characteristics of the culture to be altered and may mean that some of the plants regenerated from the culture will not be the same as the parent plant.
This subject is discussed further in Chapter 2. Cultures may also inexplicably decline in vigour after a number of passages, so that further subculture becomes impossible. In the past, it has been thought that the meristematic cells within root or shoot apices were not committed to a particular kind of development.
It is now accepted that, like the primordia of determinate organs such as leaves, apical meristems also become inherently programmed or determined into either root or shoot pathways see Chapter 8. The eventual pattern of development of both indeterminate and determinate organs is often established at a very early stage.
For example, the meristematic protrusions in a shoot apex become programmed to develop as either lateral buds or leaves after only a few cell divisions have taken place see Chapter Culture of determinate organs An organ arises from a group of meristematic cells. In an indeterminate organ, such cells are theoretically able to continue in the same pattern of growth indefinitely.
The situation is different in the primordium of a determinate organ. Here, as meristematic cells receive instructions on how to differentiate, their capacity for further division becomes limited. If the primordium of a determinate organ is excised and transferred to culture, it will sometimes continue to grow to maturity. The organ obtained in vitro may be smaller than that which would have developed on the original plant in vivo, but otherwise is likely to be normal.
The growth of determinate organs cannot be extended by subculture as growth ceases when they have reached their maximum size.
Chapter 1 Organs of limited growth potential, which have been cultured, include leaves Caponetti and Steeves, ; Caponetti, ; fruits Nitsch, , ; Street, ; stamens Rastogi and Sawhney, ; ovaries and ovules which develop and grow into embryos and flower buds of several dicotyledonous plant species Table 1. Until recently, a completely normal development was obtained in only a few cases. This was probably due to the use of media of sub-optimum composition.
By experimenting with media constituents, Berghoef and Bruinsma a obtained normal growth of Begonia franconis buds and were thus able to study the effect of plant growth substances and nutritional factors on flower development and sexual expression Berghoef and Bruinsma, b.
Similarly, by culturing dormant buds of Salix, Angrish and Nanda a,b could study the effect of bud position and the progressive influence of a resting period on the determination of meristems to become catkins and fertile flowers.
In several species, flowers have been pollinated in vitro and have then given rise to mature fruits e. Ruddat et al. Nicotiana tabacum Aquilegia formosa Cleome iberidella Nicotiana offinis Galun et al.
In some plants the production of vegetative shoots from the flower meristems on a large inflorescence can provide a convenient method of micropropagation see Chapter 2. Culture of indeterminate organs Meristem and shoot culture. The growing points of shoots can be cultured in such a way that they continue uninterrupted and organised growth.
As these shoot initials ultimately give rise to small organised shoots which can then be rooted, their culture has great practical significance for plant propagation. Two important uses have emerged: Meristem culture. Culture of the extreme tip of the shoot, is used as a technique to free plants from virus infections. Explants are dissected from either apical or lateral buds.
They comprise a very small stem apex 0. Culture of larger stem apices or lateral buds ranging from 5 or 10 mm in length to undissected buds is used as a very successful method of propagating plants. The size and relative positions of the two kinds of explant in a shoot apex of a typical dicotyledon is shown in Fig.
Node culture is an adaptation of shoot culture. Plants are propagated by dividing and reculturing the shoot clusters, or by growing individual shoots for subdivision. At a chosen stage, individual shoots or shoot clusters are rooted. Tissue cultured shoots are removed from aseptic conditions at or just before the rooting stage, and rooted plantlets are hardened off and grown normally.
Shoot culture, node culture and meristem tip culture are discussed in greater detail in Chapter 2. Embryo culture. Zygotic or seed embryos are often used advantageously as explants in plant tissue culture, for example, to initiate callus cultures.
Isolated embryo culture can assist in the rapid production of seedlings from seeds that have a protracted dormancy period, and it enables seedlings to be produced when the genotype e. During the course of evolution, natural incompatibility systems have developed which limit the types of possible sexual crosses see De Nettancourt and Devreux, The embryo, not receiving sufficient nutrition, disintegrates or aborts.
Pre-zygotic incompatibility can sometimes be overcome in the laboratory using a technique developed by Kanta et al. For a description of this technique see review articles by Ranga Swamy , Zenkteler and Yeung et al. Reviews of embryo culture have been provided by Torrey , Norstog and Raghavan , a, Embryo culture has been used successfully in a large number of plant genera to overcome postzygotic incompatibility which otherwise hampers the production of desirable hybrid seedlings.
For example, in trying to transfer insect resistance from a wild Solanum species into the aubergine, Sharma et al. Embryo culture in these circumstances is more aptly termed embryo rescue. Success rates are usually quite low and the new hybrids, particularly if they arise from remote crosses, are sometimes sterile. However, this does not matter if the plants can afterwards be propagated asexually. Hybrids between incompatible varieties of tree and soft fruits Tukey, ; Skirm, and Iris in Reuther, have been obtained by culturing fairly mature embryos.
Fruits or seeds are surface sterilised before embryo removal. Providing aseptic techniques are strictly adhered to during excision and transfer to a culture medium, the embryo itself needs no further sterilisation.
To ease the dissection of the embryo, hard seeds are soaked in water to soften them, but if softening takes more than a few hours it is advisable to re-sterilise the seed afterwards.
A dissecting microscope may be necessary to excise the embryos from small seeds as it is particularly important that the embryo should not be damaged. Culture of immature embryos pro-embryos a few days after pollination frequently results in a greater proportion of seedlings being obtained than if more mature embryos are used as explants, because incompatibility mechanisms have less time to take effect. Unfortunately dissection of very small embryos requires much skill and cannot be done rapidly: it also frequently results in damage which prevents growth in vitro.
In soybean, Hu and Sussex obtained the best in vitro growth of immature embryos if they were isolated with their suspensors intact. Excised embryos usually develop into seedlings precociously i. As an alternative to embryo culture, in some plants it has been possible to excise and culture pollinated ovaries and immature ovules.
Pro-embryos generally require a complex medium for growth, but embryos contained within the ovule require less complicated media. They are also easily removed from the plant and relatively insensitive to the physical conditions of culture Thengane et al. The difference 11 Chapter 1 between embryo and ovule culture is shown diagrammatically in Fig.
Because seedlings, which resulted from ovule culture of a Nicotiana interspecific cross all died after they had developed some true leaves, Iwai et al. Most shoots regenerated from the callus also died at an early stage, but one gave rise to a plant, which was discovered later to be a sterile hybrid. Plants were also regenerated from callus of a Pelargonium hybrid by Kato and Tokumasu The callus in this case arose directly from globular or heart-shaped zygotic embryos which were not able to grow into seedlings.
The seeds of orchids have neither functional storage organs, nor a true seed coat, so dissection of the embryo would not be possible. In fact, for commercial purposes, orchid seeds are now almost always germinated in vitro, and growth is often facilitated by taking immature seeds from green pods see Volume 2.
Many media have been especially developed for embryo culture and some were the forerunners of the media now used for general tissue culture. Commonly, mature embryos require only inorganic salts supplemented with sucrose, whereas immature embryos have an additional requirement for vitamins, amino acids, growth regulators and sometimes coconut milk or some other endosperm extract. Raghavan b encouraged the incorporation of mannitol to replace the high osmotic pressure exerted on proembryos by ovular sap.
Seedlings obtained from embryos grown in vitro are planted out and hardened off in the same manner as other plantlets raised by tissue culture Chapter 2 and Volume 2. Although embryo culture is especially useful for plant breeders, it does not lead to the rapid and large scale rates of propagation characteristic of other micropropagation techniques, and so it is not considered further in this book. Yeung et al. The induction of multiple shoots from seeds is described in Chapter 2.
Isolated root culture. Root cultures can be established from root tips taken from primary or lateral roots of many plants. Suitable explants are small sections of roots bearing a primary or lateral root meristem.
These explants may be obtained, for example, from surface sterilised seeds germinated in aseptic conditions. If the small root meristems continue normal growth on a suitable medium, they produce a root system consisting only of primary and lateral roots Fig. No organised shoot buds will be formed. The discovery that roots could be grown apart from shoot tissue was one of the first significant developments of modern tissue culture science.
Root culture initially attracted a great deal of attention from research workers and the roots of many different species of plants were cultured successfully see the comprehensive reviews of Street, , , ; and Butcher and Street, Plants fall generally into three categories with regard to the ease with which their roots can be cultured.
There are some species such as clover, Datura, tomato and Citrus, where isolated roots can be grown for long periods of time, some seemingly, indefinitely Said and Murashige, providing regular subcultures are made. In many woody species, roots have not been grown at all successfully in isolated cultures.
In other species such as pea, flax and wheat, roots can be cultured for long periods but ultimately growth declines or insufficient lateral roots are produced to provide explants for subculture. Transferring dormant meristems to fresh medium does not promote regrowth, possibly due to the accumulation of naturally-occurring auxinic growth substances at the root apex. The addition of so-called anti-auxin, or cytokinin growth regulators can often prolong active growth of root cultures, whereas placing auxins or gibberellic acid in the growth medium, causes it to cease more rapidly.
Cultures, which cannot be maintained by transferring root apices, can sometimes be continued if newly-initiated lateral root meristems are used as secondary explants instead. Liquid media are preferable, as growth in or on a solid medium is slower. This is presumably because salts are less readily available to the roots from a solidified medium and oxygen availability may be restricted.
Species, and even varieties or strains, of plants, are found to differ in their requirement for growth regulators, particularly for auxins, in the root culture medium. Isolated root cultures have been employed for a number of different research purposes. They have been particularly valuable in the study of nematode infections and provide a method by which these parasites can be cultured in aseptic conditions.
Root cultures may also be used to grow beneficial mycorrhizal fungi, and to study the process of root nodulation with nitrogen-fixing Rhizobium bacteria in leguminous plants.
For the latter purpose, various special adaptations of standard techniques have been adopted to allow roots to become established in a nitrate-free medium Raggio et al.