Plant Tissue Culture

The conventional breeding methods are the most widely used for crop improvement. But in certain situations, these methods have to be supplemented with plant tissue culture techniques either to increase their efficiency or to be able to achieve the objective, which is not possible through the conventional methods.

The term tissue culture is commonly used in a very wide sense to include in vitro culture of plant cells, tissues as well as organs. But in a strict sense, tissue culture denotes the in vitro cultivation of plant cells in an unorganised mass, e.g., callus cultures.

Another term, cell culture is used for in vitro culture of single or relatively small groups of plant cells, e.g., suspension cultures. But in general, the term tissue culture is applied to both callus and suspension cultures, and cell culture is often used for callus culture as well. When organised structures like root tips, shoot tips, embryos, etc. are cultured in vitro to obtain their development as organised structures, it is called organ cultures. In this book, the term tissue culture is used in its broad sense to denote aseptic culture of plant cells, tissues, and organs.

History of Plant Tissue Culture -

Media -

Plants in nature can synthesize their own food material. In contrast, plants growing in vitro are heterotrophic, Le., they cannot synthesize their own food material. Plant tissue culture media therefore require all essential minerals plus a carbohydrate source usually added in the form of sucrose and also other growth hormones (regulators and vitamins). Growth and morphogenesis of plant tissues in vitro are largely governed by the composition of the culture media. Although the basic requirements of cultured plant tissues are similar to those of whole plants, in practice nutritional components promoting optimal growth of a tissue under laboratory conditions may vary with respect to the particular species. Media compositions are thus formulated considering specific requirements of a particular culture system. 'For example, some tissues show better response on a solid medium while others prefer a liquid medium.

Considerable progress has been made during the past two decades on the development of media for growing plant cells, tissues and organs aseptically in culture. A significant contribution to formulation of a defined growth medium suitable for a wide range of applications was made by Murashige and Skoog (1962), In their work to adapt tobacco callus cultures for use as a hormone bioassay system, they evaluated many medium constituents to achieve optimal growth of calluses. In so doing, they improved upon existing types of plant tissue culture media to such an extent that their medium (the MS medium) has since proved to be one of the most widely used in plant tissue culture work gives the composition of different media.

Macronutrients -

The macronutrients include six major elements-nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), and surphur (S)-present as salts that constitute various media. All are essential for plant cell and tissue growth. Culture media should contain at least 25 mmoll-1 nitrate and potassium.However, considerably better results are obtained if the source for nitrogen in media is contributed by both nitrates and ammonium (2-20 mmoll-1) or any other reduced nitrogen source. In case only ammonium is used, there is need to add one or more tricarboxylic acid (TCA) cycle acids (e.g., citrate, succinate, or malate) so that any deleterious effect due to ammonium concentrations in excess of 8 mmoll-1 in the medium is diluted. When nitrate and ammonia ions are present together in the culture medium, the latter are used more rapidly. Other major elements, Ca, P, S, Mg; at concentrations in the range of 1-3 mmoll-1, appear adequate provided other requirements are satisfied.
Carbon and Energy Source -

The most preferred carbon source in plant tissue culture is sucrose. Glucose supports equally good growth while fructose is less efficient. Sucrose, while autoclaving the medium, is converted into glucose and fructose. In the process, first glucose is used and then fructose. Other carbohydrates, such as lactose, galactose, rafinose, maltose, cellobiose, melibiose, and trehalose, generally yield inferior results. Tissue cultures of Sequoia and maize endosperm can even metabolize starch as the sole carbon source.Plant cells and tissues in the culture medium lack autotrophic ability and therefore need external carbon for energy. Even tissues which are initially green or acquire green pigments under special conditions during the culture period are not autotrophs for carbon. The addition of an external carbon source to the medium enhances proliferation of cells and regeneration of green shoots.

Organic Supplements -

i) Vitamins: Plants synthesize vitamins endogenously and these are used as catalysts in various metabolic processes. When plant cells and tissues are grown in vitro, some essential vitamins are synthesized but only in suboptimal quantities. Hence it is necessary to supplement the medium with required vitamins and amino acids to achieve the best growth of the tissue. Thiamine (B1), nicotinic acid (B3), pyridoxine (B6), calcium pentothenate (B5), and myoinositol are used more often of these, thiamine is the basic vitamin required by all cells and tissues. Nicotinic acid and pyridoxine are usually added to culture media but may not be essential for cell growth in many species.Other vitamins such as biotin, folic acid, ascorbic acid, pentothenic acid, vitamin E (tocopherol), riboflavin, and p­aminobenzoic acid are also invariably used, particularly when cells are grown at very low population densities, although their requirement by plant cell or tissue cultures is apparently negligible. Generally, these vitamins are added in the range of 0.1 to 10.0 mg 1-1.

ii) Amino acids: Cultured tissues are normally capable of synthesizing the amino acids necessary for various metabolic processes. In spite of this, the addition of amino acids to media is important for stimulating cell growth in protoplast cultures and for establishing cell lines. Unlike inorganic nitrogen, amino acids are taken up more rapidly by plant cells. Casein hydrolysate (0.05-1>.1%), L-glutamine (8 mmoll-1), L-asparagine (100 mmoll-1), L-gly­cine (2 mmol,-1), 'L-arginine and L-cysteine (10 mmoll-1) are common sources of organic nitrogen used in culture media.
Tyrosine (100 mmoll-1) should be used only when agar is added to the medium. Amino acids added singly prove inhibitory to cell growth while their mixtures are frequently beneficial. Supplementing the medium with adenine sulphate can stimulate cell growth or enhance shoot formation.

iii) Other organic supplements: Culture media are often supplemented with a variety of organic extracts which have constituents of an undefined nature. These include protein (casein) hydrolysates, coconut milk, yeast and malt extracts, ground banana, orange juice, and tomato juice.In tissue culture the success achieved with the use of coconut milk (5 to 20%) and protein (casein) hydrolysates (0.05 to 1.0%) has been significant. Similarly, potato extract has been found a suitable medium for anther culture. Generally the use of natural extracts is avoided because the quantity of growth promoting constituents vary with age of the tissue from which the extract has been obtained, thereby affecting the reproducibility of results.

iv) Activated charcoal: The addition of activated charcoal (AC) to culture media is reported to stimulate growth and differentiation in orchids, carrot, ivy and tomato. Paradoxically, its effect on tobacco, soybean and Camellia has proved inhibitory. Inhibition of growth is attributed to the adsorption of phytohormones to AC, whereas stimulation could be due to any one of the factors, namely adsorption of inhibitory compounds to AC and darkening of the medium. AC is generally acid washed and neutralized before its addition at concentrations of 0.5-3.0% to the culture medium. It also helps to reduce toxicity by removing toxic compounds (e.g. phenols) produced during the culture and permits unhindered cell growth.
v) Antibiotics: Addition of antibiotics to culture media is generally avoided because their presence in the medium retards the cell or tissue growth. However, some plant cells have a systemic infection of microorganisms. To prevent the growth of these microbes it is essential to enrich the media with antibiotics. Streptomycin or kanamycin at low concentration effectively control systemic infection and media supplemented with these antibiotics do not adversely inhibit the growth of cell cultures.

Growth Regulators -

Four broad classes of growth regulators, namely auxins, cytokinins, gibberellins, and abscisic acid, are important in tissue culture. The growth, differentiation and organogenesis of tissues become feasible only on the addition of one or more of these classes of hormones to a medium. The ratio of hormones required for root or shoot induction varies considerably with the tissue, which seems directly correlated to the quantum of hormones synthesized at endogenous levels within the cells of the explant.

i) Auxins: Media are supplemented with various auxins: 1 H indole-3-a,cetic acid (IAA), 1-naphthaleneacetic acid (NAA), 1 H indole-3-butyric acid (ISA), 2,4-dichlorophenoxyacetic acid (2,4-0), and naphthoxyacetic acid (NOA). IAA occurs naturally in the plant tissues.

There are other auxins which have proven particularly effective in plant cell culture. These include 4 chlorophenoxyacetic acid (4-CPA) or p-chlorophenoxyacetic acid (PCPA), 2, 4, 5-trichlorophenoxyacetic acid (2,4,5- T), 2-methyl-4-chlorophenoxyacetic acid (MCPA), 4,amino-3,5,6-trichloropicolinic acid (picloram), and 3, 6-dichloro-2-methoxybenzoic acid (dicamba).A common feature of auxins is the property of inducing cell division. In nature the hormones of this group are involved with such activities as elongation of stem, internodes, tropism, apical dominance, abscission and rooting.

ii) Cytokinins: Cytokinins are adenine derivatives mainly concerned with cell division, modification of apical dominance and shoot differentiation in the tissue culture. The most frequently used cytokinins are 6-benzylaminopurine (SAP) or 6-benzyladenine (SA), 6+t-dimethylaminopurine or 2-N6_(P2-isopentenyl) adenine,(2-IP) N-(2-furfurylamino)-1-H-purine-6-amine (kinetin), and 6-(4-hydroxy-3-methyltrans-2-butanylamino) purine (zeatin).Zeatin and 2-IP are naturally occurring cytokinins while SA and kinetin are synthetically derived cytokinins. They are generally dissolved in dilute HCI or NaOH. It was recently discovered that N,N-diphenylurea(OPU), thidiaziron, N-2­chloro-4-puridyl-N-phenylurea (CPPU), and other derivatives of diphenylurea show cytokinin type activity.

The ratio of auxins and cytokinins is important with respect to morphogenesis in the culture system. For embryogenesis, callus initiation, and root initiation the requisite ratio of auxins to cytokinin is high, while the reverse leads to axillary and shoot proliferation. The mechanism of cytokinin action is not clearly understood although there are reports about the presence of compounds with cytokinin activity in transfer RNA (tRNA). Cytokinins have also been shown to activate RNA synthesis and to stimulate protein and enzyme activity in certain tissues.

iii) Gibberellins and Abscisic Acid: These growth compounds are occasionally used in tissue culture. In some species these hormones are required to enhance and in others to inhibit growth. GA is the .most common gibberellin used of the over 20 known ones. It promotes the growth of cell cultures at low density, enhances callus growth and induces dwarf or stunted plantlets to elongate. Abscisic acid (ASA) in the culture medium either stimulates or inhibits callus growth depending on the species.

Solidifying OR Gelling Agents -

Gelling or solidifying agents are commonly used for preparing semisolid or solid tissue culture media. In static liquid cultures the tissue or cells become submerged and die due to lack of oxygen. Gels provide a support to tissues growing in static conditions. Agar, a polysaccharide obtained from seaweed, has several advantages over other gelling agents. First, agar gels do not react with media constituents. Secondly, they are not digested by plant enzymes and remain stable at all feasible incubation temperatures. Normally, 0.5 to 1 % agar is used in the medium to form a firm gel at the pH typical of plant cell culture media. In nutritional studies the use of agar is avoided because commercially available agar contains impurities in the form of Ca, Mg, K, Na, and trace elements. Consequently, a change in the agar concentration also affects the nutrients present in it as well as the overall nutrient concentration in the experiment.

Impurities can be removed, however, for such critical experiments by washing agar in double distilled water for at least 24 hr, rinsing in ethanol and drying at 600 C for 24 hr.

Gelatin at high concentrations (10%) has been tried as a gelling agent but has limited use because it melts at low temperature (250 C). Other compounds successfully tested include biogel (polyacrylamide pellets) alginate, phytagel and Gelrite.

The FMC Corporation has recently developed a highly purified agarose called Sea Plaque(k), which can be used in the recovery of single protoplasts from cultures. Perforated cellophane, filter paper bridges, filter paper wicks, polyurethane foam and polyester fleece are alternative methods of support used in the medium for cell or tissue growth.

The advantage of working with synthetic gelling compounds is that they form clear gels at relatively low concentrations (1.25-2.5 g 1-1) and are valuable aids for detecting contamination that may develop during the span of cultures. Whether explants grow better on agar or other supporting agents depends on the tissue and the species. pH: Plant cells and tissues require optimum pH for growth and development in cultures. While preparing a medium, the pH can be adjusted to the requirement of an experiment.

The pH affects uptake of ions and for most of the culture media pH 5.0 to 6.0 before sterilisation is considered optimal.Higher pH is likely to give a hard medium while a low pH results in unsatisfactory solidification of the agar. Most tissue culture media are poorly buffered, resulting in pH fluctuations which could prove detrimental to long term survival and growth of single cells or cell populations at low density.

Media Preparation -

Media making can be time consuming. Nowadays the plant tissue culture media most commonly used are available in the market as dry powders. The simplest method of preparing media is to dissolve these powders containing inorganic and organic nutrients in some quantity of distilled water. After the contents have been thoroughly mixed in water, sugar, agar (melted), and other organic supplements are added. Finally, the volume is made up to one litre. The pH is adjusted and the medium is autoclaved. Powdered media are useful for propagation of plant species requiring nutrients according to the recipe of standard media.

In experiments in which changes in the quantity and quality of media constituents become necessary, it is desirable to weigh and dissolve each ingredient separately before mixing them together. Another convenient procedure is to prepare stock solutions which, when mixed together in appropriate quantities, constitute a basal medium. Mistakes in the preparation of media can do greater harm than any other fault in the tissue culture technique. It is absolutely essential that all steps in the media preparation and composition be followed carefully.

Nutrient Media -

Virtually all tissue culture media are synthetic or chemically defined; only a few of them use complex organics, e.g., potato extract, as their normal constituents. A synthetic medium consists of only chemically defined compounds. A variety of recipes have been developed since none of them is suitable for either all plant species or for every purpose. Most of these recipes have been elaborated from those of White (itself evolved from a medium for algae) and Gautheret (based on Knop's salt solution). The composition of White, MS and B5 (the last two are the most commonly used) media.

Composition of Some Plant Tissue Culture Media -

Gelling agent (for callus cultures) -

Agar
Generally 6-8 g/l of the mediumMedium pH generally adjusted to 1N HCl or 1N NaOH

Callus and Suspension Cultures -

When explants are cultured on a suitable GR combination, many of its cells undergo division. Even mature and certain differentiated: e.g., parenchyma and often colenchyma, cells undergo changes to become meristematic; this is called dedifferentiation. Dedifferentiation involves, among other things, renewed and enhanced RNA and protein syntheses leading to the formation of new cellular components needed for meristematic activity. Initially, cell division is confined to the cut ends, but subsequently it covers the entire explant. The resulting cell mass is ordinarily unorganised, but it often consists of several cell types including fibers, and vascular elements.
Callus Cultures - Tissues and cells cultured on an agar gelled medium form an unorganised mass of cells called callus. Callus cultures need to be subcultured every 3-5 weeks due to cell growth, nutrient depletion and medium drying. Calluses are easy to maintain and are the most widely used

Suspension Cultures -

Tissues and cells cultured in a liquid medium produce a suspension of single cells and cells clumps of few to many cells: these are called suspension cultures. Liquid cultures must be constantly agitated, generally by a gyratory shaker at 100-250 rpm (revolution per minute), to facilitate aeration and dissociation of cell clumps into smaller pieces. Suspension cultures grow much faster than callus cultures, need to be subcultured about every week, allow a more accurate determination of the nutritional requirements of cells and are amenable to scaling up for a large scale production of cells and even somatic embryos (SEs). The suspension cultures are broadly grouped as:

(1) batch cultures,

(2) continuous cultures, and

(3) immobilized cell cultures.

Batch Cultures -

In a batch culture the same medium and all the cells produced are retained in the culture vessel, e.g., culture flasks (100-250 ml), fermenters (variable size), etc. The cell number or biomass of a batch culture exhibits a typical sigmoidal curve, having a lag phase during which the cell number remains unchanged, followed by a logarithmic (log) phase when there is a rapid increase in cell number and finally ending in a stationary phase during which cell number does not change. The lag phase duration depends mainly on inoculum size and growth phase of the culture from which inoculum is taken. The log phase lasts about 3-4 cell generations, and the duration of a cell generation (time taken for doubling of cell number) may vary from 22-48 hr mainly depending on the plant species.

The stationary phase is forced on the culture by a depletion of the nutrients and possibly due to an accumulation of cellular wastes. If the culture is kept in stationary phase for a prolonged period the cells may die. Batch cultures are maintained by subculturing. They are used for initiation of cell suspensions, which may be used for cloning, cell selection or as seed cultures for scaling up or for continuous cultures. They are, however, unsuitable for studies on cell growth and metabolism because there is a constant change in cell density and nutritional status of the medium. But batch cultures are much more convenient than continuous cultures and, as a result, are routinely used.

Continous Cultures -

In a continuous culture, the cell population is maintained in a steady state by regularly replacing a portion of the used or spent medium by fresh medium. Such culture systems are of either(1) closed or(2) open type. In a closed continuous culture, cells are separated from the used medium taken out for replacement, and added back to the culture so that cell biomass keeps on increasing. In contrast, both cells and the used medium are taken out from open continuous cultures and replaced by equal volume of fresh medium.

The replacement volume is so adjusted that cultures remain at submaximal growth indefinitely. The open cultures are of either turbidostat or chemostat types. In a turbidostat, cells are allowed to grow up to a preselected turbidity (usually measured as OD) when a predetermined volume of the culture is replaced by fresh normal culture medium.

But in a chemostat, a chosen nutrient is kept in a concentration so that it is depleted very rapidly to become growth limiting, while other nutrients are still in concentrations higher than required. In such a situation, any addition of the growth limiting nutrient is reflected in cell growth. Chemostats are ideal for the determination of effects of individual nutrients on cell growth and metabolism.

Immobilized Cell Cultures -

Plant cells and cell groups may be encapsulated in a suitable material, e.g., agarose, calcium alginate, etc., or entrapped in membranes or stainless steel screens. The gel beads containing cells may be packed in a suitable column or, alternatively, cells may be packed in a column of a membrane or wire cloth. Liquid medium is continuously run through the column to provide nutrients and aeration to cells. Immobilization of cells changes their cellular physiology in comearison to suspension culture cells; this offers several advantages for their use in biochemical production, but they are usually not used for other studies

Subculture -

After a period of time, it becomes necessary, chiefly due to nutrient depletion and medium drying, to transfer organs and tissues to fresh media. This is particularly true of tissue and cell cultures where a portion of tissue is used to inoculate new culture tubes or flasks; this is known as subculturing.In general, callus cultures are subcultured every 4-6 weeks, while suspension cultures need to be subcultured every 3- I 4 days. Plant cell and tissue cultures may be maintained indefini1iely by serial subculturing. In case of suspension cultures, subculturing should be done about or somewhat prior to the time of their maximum growth. The inoculum volume should be 20-25% of the fresh medium volume; in any case, the initial cell density of the fresh culture (just after inoculation) should be 5 x 104 cells ml or higher otherwise the cells may fail to divide.

Estimation of Cell Growth -

Cell number is the most informative measure of cell growth. This measurement is applicable to only suspension cultures, and even there cell aggregates must be treated, e.g., with pectinase, to dissociate them into single cells before counting the cell number in a haemocytometer.

Therefore, cell number is estimated only where information obtained justifies the efforts.In contrast, packed cell volume of suspension cultures is easily determined by pipetting a known volume into a 15 ml graduated centrifuge tube, spinning at 200 x g for 5 min and reading the volume of cell pellet, which is expressed as ml cells of culture

Culture fresh and dry weights are the most commonly used measures of growth of both suspension and callus cultures. In case of callus cultures, the cell mass is placed on a preweighed dry filter paper or nylon filter and weighed to determine fresh weight. Cells from suspension cultures are filtered onto a filter paper or nylon filter, washed with distilled water, excess water removed under vacuum and weighed alongwith the filter; the filter is preweighed in wet condition. For dry weight determination the cells and the filter are dried in an oven at 60°C for 12 hr and weighed; the filter is pre-weighed in dry condition. Cell fresh and dry weights may either be expressed as per ml (suspension culture) or per culture.

Nuclear Cytology -

Callus and suspension cultures show both numerical (polyploidy and aneuploidy) and structural (deletions, translocations, etc.) chromosome changes. The frequency of these changes tends to increase with time so that some cultures may become predominantly or even completely polyploid or aneuploid. Explants contain endopolyploid cells, which may give rise to a portion of polyploid cells in cultures. But most polyploid cells appear to originate through endoreduplication (additional rounds of DNA replication without intervening cell division) although selection for such cells can not be ruled out.

Aneuploid cells originate mainly due to anaphase irregularities like unequal chromatid separation, lagging chromatids or chromosomes, anaphase bridges giving rise to breakage-fusion-bridge cycle, chromosome fragmentation, etc. The cytogenetic status of cultured cells is influenced by several factors of the culture system, e.g., GR concentrations and combination, culture age, liquid or agar medium, subculture interval, sucrose concentration, etc. Suspension cultures of many diploid species show a selection for diploid cells so that they remain predominantly diploid for long periods, e.g., Vida hajastana and Haplopappus gracilis cultures for over 300 days.

Cloning -

A clone of cells consists of all the cells derived through mitosis from a single cell. and the process of obtaining a clone is called cloning. Therefore, all the cells of a clone are expected be identical with each other in their genotype and karyotype (chromosome constitution), and other attributes, except for the changes that may arise afresh during and after cloning. Cloning is based on single cells separated from tissues and cultured in a manner to allow separate recovery of the cell mass derived from them.
Cell Viability Test - Cell viability can be determined by any one of the following approaches: (1) phase contrast microscopy, and staining with (2) 2, 3. 5-triphenyltetrazolium chloride (TIC), (3) fluorescein diacetate (FDA) and (4) Evan's blue.Live cells show cytoplasmic streaming and a well defined healthy nucleus which are easily observable with a phase contrast microscope or even a light microscope. Cell masses can be stained with 1-2% solution of TTC, which is reduced by living cells to formazan that yields red colour.

Formazan can be extracted and measured with a spectrophotometer to give a quantitative estimate of viability, but it is not suitable for single or few cells. Cell are treated with 0.01 % solution of FDA. Live cells cleave FDA by esterase activity and produce fluorescein, which can not cross plasma membrane. With UV exposure, fluorescein gives green fluorescence so that live cells appear green, while dead cells do not fluoresce. Evan's blue (0.025%) is not taken up by live cells, while it freely enters into damaged dead cells. Therefore all cells that take up stain are dead. Evan's blue is usually used in conjunction with FDA.

Regeneration in Tissue Culture -

Regeneration refers to the development of organised structures like roots, shoots, flower buds, somatic embryos (SEs), etc. from cultured cells/tissues; organogenesis is also used to describe these events. Root regeneration occurs quite frequently, but it is useful only in conjunction with shoots and embryo germination. Only shoot and SE regenerations give rise to complete plants, which is essential for applications of tissue culture technology in agriculture and horticulture. Often differentiation is used as synonym for regeneration. But differentiation describes the development of different cell types, e.g., vascular elements, etc., as well (cyto-differentiation). Therefore, it is more appropriate to use phrases like shoot differentiation, SE differentiation, etc. than using the term differentiation alone. Regeneration may occur either directly from the explant or may follow an intervening callus phase.

Shoot Regeneration -

Cultured cells of many plant species show shoot regeneration under appropriate conditions. Shoot buds usually arise from a group of meristematic cells called meristemoids, which give rise to leaf primordia and the apical meristem. The developing buds develop procambial strands, which become connected with the pre-existing vascular tissue present in the explant/callus. In contrast; a somatic embryo (SE) has no such vascular connection with the explant/callus and, as a result, is easily separable from it.When callus is transferred onto a medium favouring shoot regeneration, clusters of meristematic cells, called nodules or meristemoids appear. Meristemoids are considered to arise in areas that accumulate starch, which is believed to serve as an energy source for shoot bud differentiation.

GA3 is thought to inhibit shoot regeneration by interfering with starch accumulation. Meristemoids may develop vascular elements inside them, while their outside may be made of cambium like cells. Initially, the meristemoids may either produce a root or shoot. In general, roots originate from inside the meristemoids (endogenous origin), while shoots develop from the outside (exogenous origin), but in some cases shoots originate endogenously. Shoot regeneration is affected by a number of factors.

Growth Regulators and Other Factors for Shoot Regeneration -

In general, shoot regeneration is promoted by cytokinins, while auxins seem to have an inhibitory effect. The classical studies of Skoog and coworkers on shoot regeneration from tobacco pith culminated in the well known postulate that organ formation depended on the auxin/cytokinin ratio and not on their absolute concentrations. In tobacco a higher ratio of cytokinin led to shoot regeneration, while a higher ratio of auxin promoted root regeneration.The auxin/cytokinin ratio concept does not seem to apply equally well to other species.

(1) In some species, e.g., Convolvulus, Bacopa, Citrus, shoot regeneration occurs on GR-free media, but is promoted by a cytokinin.

(2) In some other cases, e.g., chickpea immature cotyledons, shoot regeneration occurs only when cytokinin is provided; presence of a low concentration of auxin may have a promotory effect.

(3) In plants like Cyclamen, auxin concentration alone determined whether a shoot or root would regenerate; in contrast, adenine (a cytokinin) affects only the frequency of their regeneration.

(4) Alfalfa presents a peculiar case: callus is first initiated on a 2, 4-0 and kinetin containing medium; shoot or root regeneration occurs when it is subcultured on a GR-free medium.

The ratio of auxin to kinetin in the first medium determines the type of organ formed: a high 2, 4-0 concentration results in shoot regeneration, while a high kinetin level supports root regeneration.

In general, GA3 inhibits shoot bud regeneration. But in some species, e.g., Chrysanthemum, Arabidopsis, GA3 promotes shoot regeneration. In contrast, a large number of species show enhanced shoot regeneration due to ABA (abscisic acid), which counteracts many effects of GA3.The variable responses of different plant species to the exogenous GRs may be explained as follows. Shoot and root regeneration require specific levels of the different growth hormones, viz., auxin, cytokinin, and gibberellin. However, the endogenous levels of these hormones may vary considerably among different plant species so that a hormone may be either suboptimal, optimal or supraoptimal for shoot (or root) regeneration. The response of a plant species to an exogenous GR would, therefore, depend mainly on the endogenous level of that GR (and of other GRs as well) in that species.

Thus it has been postulated that in species like Chrysanthemum, GA3 occurs in suboptimal concentrations so that exogenous GA3 has a promotory effect. But in those species where its endogenous concentration is supraoptimal, ABA promotes shoot regeneration. It may be added that in some species auxin inhibitors enhance shoot bud differentiation.Other Factors. Shoot regeneration is markedly affected by the genotype of explant in that different varieties of a given species show quite different frequencies of shoot regeneration. In alfalfa, breeding and selection drastically increased regeneration ability. In wheat, callus growth and regeneration ability are governed by genes called, tissue culture response (TCR) genes, which have been mapped on specific chromosomes.In addition, physical condition of medium (liquid or agar) has a marked influence on shoot regeneration; in some cases liquid medium was superior, while in others it was drastically inferior to agar medium. Light seems to have an inhibitory effect, and even the quality of light may be important. The optimum temperature for shoot regeneration may vary with the plant species.

Somatic Embryogenesis -

A somatic embryo (SE) is an embryo derived from a somatic cell, other than zygote, usually on culture in vitro, and the process is known as somatic embryogenesis. In contrast, embryos developing from zygotes are called zygotic embryos or often simply embryos, while those derived from pollen are known as pollen embryos or androgenetic embryos. By 1993, somatic embryogenesis was reported from over 100 species.Developmental Pattern of SEs. SEs generally originate from single cells, which divide to form a group of meristematic cells.

Usually, this multicellular group becomes isolated by breaking cytoplasmic connections with the other cells around it and subsequently by cutinization of the outer walls of this differentiating cell mass.The cells of meristematic mass continue to divide to give rise to globular (round ball-shaped), heart-shaped, torpedo and cotyeledonary stages. In general, the essential features of SE development, especially after the globular stage, are comparable to those of zygotic embryos.
Somatic embryos are bipolar structures in that they have a radicle and a plumule. The radicular end is always oriented toward the centre of callus or cell mass, while the plumular end always sticks out from the cell mass. In contrast, a shoot bud is monopolar as it does not have a radicular end.

In many SEs, radicle is suppressed so that they often do not produce roots; in such cases, roots have to be regenerated from the shoots produced by germinating SEs. SEs often show abnormal developmental features, e.g., 3 or more cotyledons, bell shaped cotyledon, larger size etc.; these problems are often overcome by the presence of ABA or a suitable concentration of mannitol.In some species, normal looking somatic embryos are formed, but they fail to germinate.

Somatic embryogenesis is influenced by several factors, e.g.

(1) GRs

(2) nitrogen source,

(3) explant,

(4) genotype and

(5) others

Finally, transformed cells produce opines, such as octopine, and nopaline, chemicals formed from two amino acids. Arginine and alanine form octopine while arginine and glutamine produce nopaline. Octopine and nopaline are not found in normal plant cells.

There are two more or less distinct types of crown galls. The first type grows as a relatively unorganized callus on artificial medium and on host plants. Cells of this type produce octopine. The second type grows as a callus containing green islands. The islands show a variable amount of organization, inducing production of multiple shoots. Cells of this type produce nopaline. The two types are associated with different strains of A. tumefaciens. There is also a related disease ("hairy root disease") in which infected tissues proliferate in root tissue and produce an opine. This disease is associated with the bacterium A. rhizogenes.Formation of opines explains the ecological significance of tumor formation. Each strain of Agrobacterium synthesizes enzymes (permease, dehydrogenase) that allow it to metabolize the specific type of opine formed by the tumor it induces. Thus by stimulating the plant to form opines, the bacteria insure themselves a supply of nutrients specifically designed for them. Growth of the infected tissue is important because it increases the amount of opine forming tissue. It is possible, too, that the physical characteristics of the tumors parenchymal cells with extensive intercellular spaces provide a good habitat for the bacteria. Early studies of the mechanism of "transformation" demonstrated that tumorous tissue remained transformed even in the absence of infecting bacteria. (The bacteria could be removed with antibiotics such as penicillin.).

Growth Regulators and Other Factors for Somatic Embryogenesis -

In most species an auxin (generally 2,4-0 at 0.5-5 mg/l) is essential for somatic embryogenesis. The auxin causes dedifferentiation of a proportion of cells of the explant, which begin to divide. In carrot, these small, compact cells divide asymmetrically, and their daughter cells stick together to produce cell masses called pro embryogenic masses or embryogenic clumps (ECs). In the presence of auxin, the ECs grow and break up into smaller cell masses, which again produce ECs. But when the auxin is either removed or reduced (0.01-0.1 mg/l) and cell density is lowered, each EC gives rise to few to several SEs; each SE is believed to develop from a single superficial cell.

The ability to regenerate SEs, i.e., totipotency, is acquired by cells during dedifferentiation in response to high auxin treatment but the mechanism is not well known. Some glycoproteins produced by totipotent cell masses are secreted into the medium; when these proteins are added into the culture medium they speed up the process of acquisition of totipotency. A class of proteins, called arabinogalactan proteins, induces SE regeneration in undifferentiated carrot cells, indicating their role in the process. Auxins promote hypermethylation of DNA which may have a role in totipotency acquisition. In many species like carrot, coffee; alfalfa etc., somatic embryogenesis is a two step process:

(i) SE induction on high auxin (up to 40-60 mg/l 2, 4­ D) and

(ii) SE development on a low auxin or OR-free medium.

In the SE induction phase, explant cells dedifferentiate, became totipotent and, in many species, form embryogenic clumps (ECs). Cells can be maintained in embryogenic stage on the induction medium for prolonged periods (over 10 years in carrot). When ECs are transferred from induction medium to an appropriate medium, SE differentiation proceeds from globular, heart-shaped, torpedo to cotyledonary stages; this is called SE development phase. Clearly in species like carrot, etc., OR requirements for the two phases are drastically different. In most cases, SEs begin to germinate immediately after the cotyeledonary stage; this is called SE conversion. But often the plantlets so obtained are rather weak. It is, therefore, desirable to subject SEs to a maturation phase, following their development; in this phase the SEs usually do not grow but undergo biochemical changes to become more sturdy and hardy.

SE maturation is achieved by culturing than on a high sucrose (up to 6% or even 40%) medium or in presence of a suitable concentration (0.2-0.4 mg/l) of ABA, or by subjecting them to partial desiccation. In most species, SE maturation improves their conversion, often by several-fold. In some species, e.g., Cicer arietinum, wheat etc., SE induction and development may take place on the same high auxin medium, although the frequency of mature embryos is rather low.

In some species, SEs are produced in response to a cytokinin, e.g., BAP induces SEs in hypocotyls of young zygotic embryos of Trifolium sp., pea, etc. But SEs are produced on immature cotyledons of these explants when 2,4-D is used in the .medium. It seems that cytokinins are effective in SE regeneration from embryogenic cells of young zygotic embryos, while auxins are effective on differentiated cells of both embryos and somatic tissues. Many workers have used combinations of auxins and cytokinins for SE regeneration in different species, but the role of cytokinin in these systems is not known.

Other FactorsCertain other factors are reported to affect SE regeneration. For example, high K+ levels and low dissolved O2 levels promote SE regeneration in some species. In some other species, e.g. Citrus medica, some volatile compounds like ethanol inhibit SE regeneration. In soybean, low sucrose concentrations (5 and 10 g/l) promote SE regeneration as compared to high concentrations (20 and 30 g/l). In alfalfa, use of maltose as carbon source improves both SE induction and maturation (including germination) as compared to those on sucrose.

Comparison of Shoot Buds and Somatic Embryos -

Nitrogen Source for Somatic Embryogenesis -
The form of nitrogen has a marked effect on somatic embryogenesis. In carrot, NH+4 has a promotive effect on SE regeneration. In fact, induction of SEs in carrot occurs only when about 5 m mol/kg of cell fresh weight NH+ * is present in the cells. This level of endogenous NH+ 4 is reached with only 2.5 m mol/l of exogenous level of NH+4, while 60 m mol/l NO-3 is needed for the same. Therefore, the presence of a low level of NH+4(in carrot 10 m mol/l is optimal) in combination with NO-3 is required for SE regeneration. In carrot,NH+4 is essential during SE induction, while SE development occurs on a medium containing NO-3 as the sole nitrogen source.


Genotype of Explant in Somatic Embryo Regeneration - Explant genotype has a marked influence on SE regeneration, and in many cases it may determine whether or not SE regeneration will occur. Strong genotypic effects have been shown in many species, e.g., alfalfa, wheat, maize, rice, chickpea, etc. In case of alfalfa, individual genes affecting SE regeneration have been identified.

In case of wheat, chromosome 4B is important in regeneration, a major gene affecting regeneration is located on the long arm of chromosome 2D, minor genes on the long arm of chromosome 2A and short arm of 2B, and a regulatory gene on the long arm of chromosome 2B.

Variation for regeneration ability is mainly additive and highly heritable in maize, rice and wheat, but in barley dominance seems to be more important. In case of wheat, rice and maize cytoplasm has a strong influence on regeneration.

Anthur Culture -
Haploid plants may be obtained from pollen grains by placing anthers or isolated pollen grains on a suitable culture medium; this constitutes anther and pollen culture, respectively. The anthers may be taken from plants grown in the field or in pots, but ideally these plants should be grown under controlled temperature, light and humidity; the optimum conditions may differ from species to species. Often, the capacity for haploid production declines with the age of donor plants. Flower buds of the appropriate developmented stage are collected, surface sterilized, and their anthers are excised and placed horizontally on culture medium.

Some workers prefer to partially embed the anthers in the culture medium. Flower buds with small anthers may them selves be cultured and, in some cases, the entire inflorescence has been cultured. Care should be taken to avoid injury to anthers since it may induce callus formation from anther walls.

Alternatively, pollen grains may be separated from anthers and cultured on a suitable medium. In many plant species, SEs from the Pollen grains of cultured anthers are¬ directly produced, e.g., in Datura; Atropa, Brassica campestris, B. napus, several Nicotiana sp. (including N. tabacum and N. rustica), Petunia axillaris, etc

In such cases, the plants obtained from germination of embryos are generally haploid, but some polyploids are also produced. But in many other species like rice (O. vulgare), barley (H. vulgare), wheat, tomato, triticale, etc. pollen grains produce callus from which plantlets may be regenerated under suitable culture conditions. In these cases, the ploidy level of plants varies considerably more than in those where embryos are produced. Haploid plantlets have been regenerated from pollen grains of about 200 species of over 50 genera and 25 families. Of these, the following are examples of important crop species: potato (S. tuberosum), barley, wheat (Triticum sp.), rice, Brassica campestris, Triticale, many members of Solanaceae and some vegetables.

Pathways of Development in Pollen Grains -

The early divisions in responding pollen grains may occur in one of the following four ways.

(i) The unicleate pollen grain may divide symmetrically to yield two equal daughter cells both of which undergo further divisions, e.g., Datura innoxia (Pathway I).

(ii) In some other cases, e.g., N. tabacum, Datura metel, barley, wheat, triticale; chillies, etc., the unicleate pollen divides unequally (as it does in nature). The generative cell degenerates callus/embryo originates due to successive divisions of the vegetative cell (Pathway. II).

(iii) But in few species, e.g., Hyoscyamus niger, the pollen embryos originate from the generative cell alone; the vegetative cell either does not divide or divides only to a limited extent forming a suspensor like structure (Pathway III).

(iv) Finally, in some species, e.g. Datura innoxia, the uninucleate pollen grains divide unequally, producing generative and vegetative cells, but both these cells divide repeatedly to contribute to the developing embryo/callus (Pathway IV).

Most of the pollen grains of many crop species, e.g., tobacco, barley, wheat, etc., are bigger, stain deeply with acetocarmine and contain plenty of starch. But a small proportion (Ca. 0.7%) of the pollen grains are smaller and stain faintly with acetocarmine; these are called S-grains.

This phenomenon is known as pollen dimorphism. It is these S-grains, which respond during anther culture; the frequency of responding pollen grains can be enhanced over that of S-grains by certain pretreatments, e.g., chilling.

Pollen grains of the cultured anthers show remarkable cytological changes during the first 6-12 days, called the inductive period. In tobacco, the gametophytic cytoplasm of binucleate pollen grains is degraded, ribosomes are eliminated and only few mitochondria and plastids remain. New ribosomes are synthesized following the first sporophytic division of the vegetative cell.

The responsive pollen grains become multicellular and ultimately burst open to release the cell mass. This cell mass may either assume the shape of a globular embryo and undergo the developmental stages of embryogeny, or it may develop into a callus depending on the plant species.

In some species, e.g., rice, wheat, rye, maize, etc., the pollen grains can be induced to produce embryos or calli by simply altering the medium composition. Careful studies in tobacco have shown that

(i) over 80% of well developed embryos are associated with their radicular ends to the anther wall and that

(ii) the exine of pollen grains must rupture in such a way as to expose the putative plumular ends of the developing cell mass for their further differentiation into SEs.

The latter ensures that the exine lies between the would be radicular end and the supporting tissue to which the cell mass is adhered; this is believed to be important in establishing polarity, which appears to be essential for SE differentiation.

Culture Medium for Pollen Embryogenesis -
Medium requirements may vary with the species, the genotype, the age of donor plants and anthers, and the conditions under which the donor plants are grown. For example, pollen grains of Datura and tobacco produce embryos on an agar medium containing only 2-4% sucrose, while elaborate media e.g., N6 and Potato-2 media, had to be formulated for cereals. Sucrose is essential for anther cultures; the concentration may range from 3.% for barely to 6% for wheat and potato, but 2-3% sucrose is most commonly used. For most plant species, a complete tissue culture medium is required; MS, LS (Linsmaer and Skoog) and some other tissue culture media are generally used.

Media with dilute salt solutions, e.g., White's and Heller's media, are ordinarily supplemented with coconut milk. Sucrose plays a key role in the induction of embryogenesis, while other medium constituents appear to be needed for post induction development of embryos. High sucrose level may +play an osmo regulatory role during induction, but it is not necessary, or even detrimental, during embryo development.

Growth Regulators for Pollen Embryogenesis - In Solanaceous plants, pollen embryogenesis does not require any growth regulators, but low levels of auxins, cytokinins and even GA3 appear beneficial; 0.1 mg/l IAA gave the best results. In Hyoscyamus niger an auxin, e.g., 2 mg/l 2, 4-D, enhanced the frequency of responding calli but had no effect on the number of embryogenic pollen grains.

In contrast, cytokinins (0.01-10 mg/l) reduced the number of pollen grains producing embryos most likely by interfering with cell division in induced pollen grains. In species where callus is formed, e.g., cereals, auxins and cytokinins are almost invariably used either in combination or in sequence, but the role played by them is not known. It seems that different GRs may be required for best results with different plant species.

The presence of an auxin may determine the mode of subsequent development of androgenic cell masses. Wheat anthers cultured on a medium having 2, 4-D produce callus, while those kept a coconut milk supplemented medium give rise to embryos.

Similarly, when anthers of indica rice are cultured in the presence of an auxin, pollen grains begin to develop embryos, which continue in this mode if the anthers are transferred to an auxin-free medium. But if they are left on the auxin-supplemented medium, callus is produced.

Stage of Pollen Development -
The optimum stage of pollen varies with the species. For many species, including Datura, tobacco, etc., the optimum stage is just before or just after the first pollen mitosis, while the early binucleate stage is the most suitable for species like Atropa belladona and Nicotiana sylvestris, and is absolutely essential for Nicotiana knightiana. In cereals, the best stage appears to be the early or mid uninucleate stage, i.e., before the first pollen mitosis. In contrast, in species like tomato and Arabidopsis thaliana, the optimum stage is when the PMC's are in meiosis I, while in Brassica trinucleate pollen grains (at the time of pollen shedding) are the best.

Pollen grains of Brassica remain responsive throughout the maturation phase, but their auxin requirement increases with the pollen age.In tobacco, the beginning of starch accumulation in pollen grains marks the end of their embryogenic potential. Further, the presence of starch in early binucleate pollen is indicative of a lack of androgenic potential of the species, while in species having the potential starch is absent.

Culture Environment -
Anther cultures are generally maintained in alternating periods of light (12¬18 hr; 5,000-10,000 lux m2) at 28°C and darkness (12-6 hr) at 22°C, but the optimum conditions vary with the species. The walls of responsive anthers turn brown and after 3-8 weeks they burst open due to the developing callus or embryos. After the seedlings (from embryos) or shoots (from callus) become 3-5 cm long, they are transferred to a medium conducive to good root development. Finally, they are transferred to soil in the same way as other in vitro regenerated plantlets.

In tobacco, optimum temperature is around 25°C. Pollen embryos are not formed in D. innoxia anthers cultured at 20°C or below. Clearly temperature optimum varies with the species. In some species, e.g., grape, potato, Datura, etc., exposure of anthers to light during their first 24 hr of culture enhances the frequency of haploid callus or responding anthers. Light appears to be beneficial even in those species where anthers cultured in dark respond adequately. Isolated pollen grains require a relatively low intensity of light.

Pretreatment of Flower Buds -
Exposure of excised flower buds to a low temperature for some time, e.g., at 3-5°C for 2 days or at 7-8°C for 12 days for tobacco, prior to removal of anthers for culture may markedly enhal1ce the recovery of haploid plants . In some species, however, a brief exposure of anthers to a high temperature is reported to have a promotory effect, e.g., at 35°C for 24 h in Brassica campestris. It contrast, the best pretreatment for cereals like wheat, rice, barley, etc. seems to be 3-28 days at 4-10°C; this increases the frequency of green plants. The pretreatment temperature and duration may be considerably affected by plant species, genotype and stage of anther development.

List of Stress Treatment That Induce Androgenesis -
 

Other Factors Affecting Androgenesis -

Androgenesis in barley is promoted by the use of wheat ar barley starch as gelling agent (in place of agar), and by the addition of Ficoll in liquid medium. In many species, activated charcoal (in agar-gelled media) is promotive. In addition, amino acids like glutamine, praline, serine, etc. enhance the frequency of responsive anthers. Anther extracts and media conditioned by culturing anthers far few days improve andragenesis; thus anther wall seems to provide same nutritional factors.

Pollen Culture -
Isolated pollen grains, when cultured in vitro, give rise to haploid embryos ar callus; this approach is called pollen culture. Pollen may be isolated either by squeezing ar float culturing the anthers.

About 50 anthers may be placed in 20 ml of medium and squeezed with a glass rod; the solution is filtered through a nylon mesh of suitable pare size (25 µm far tomato to 100 µm far maize), and centrifuged at 500-800 rpm far 5 min.

The pollen pellet is collected, washed twice and. suspended at a final density of 103-104 pollen/ ml. In float culture, excised anthers are floated a shallow liquid medium in Petri dishes; the anthers dehisce in a few days releasing their pollen grains into the medium.

These anthers continue to shed pollen so that their serial subculture yields pollen samples in different stages of andragenesis (haploid embryo/plantlet formation from pollen grains).Initially, isolated pollen grains were cultured either in hanging drops or on a filter paper raft placed on cultured anthers.

Subsequently, Nitsch and coworkers first replaced the nurse tissue by an extract of cultured anthers, and finally devised a completely synthetic medium far pollen culture the crucial ingredients of which were glutamine, L-serine and inositol. Successful pollen culture is reported far several species, e.g., tobacco, Datura, petunia, potato, barley, wheat, rice, maize, rapeseed, etc.

Pollen culture offers certain advantages aver anther culture due to the elimination of anther wall, e.g.,

(i) studies an differentiation and development are easier and mare precise,

(ii) no. callus formation can occur from wall tissue and

(iii) products from different pollen grains ordinarily do not get mixed up (this eliminates the risk of chimaera).

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