Primary Growth Requirements

Plants Primary Growth

Primary growth is defined as the processes in a plant that are essential for the growth of the meristematic regions such as the shoot apex, root tip, and axillary meristems.

Plant cell and tissue cultures have specific optima for their primary growth in terms of lighting, temperature, aeration, a nutrient medium that must supply a carbon source, vitamins, hormones, and inorganic constituents and with pH typically between 5.5 and 6.5. Aeration can be critical depending upon the species.

Table of Typical Product of Plants

Although a few exceptions do exist where glucose, fructose, or galactose is preferred, the majority of the plant cultures use sucrose. Usual trace requirements are thiamine, niacin, riboflavin, pyridoxine, choline, ascorbic acid, and inositol. Hormones such as auxins and cytokinins promote an undifferentiated state or trigger differentiation into specific plant tissues. As with the whole plant, cellular groups, either differentiated or undifferentiated, require a set of inorganic elements such as nitrogen, phosphorus, potassium, magnesium, calcium, sulfur, iron, chlorine, boron, manganese, and zinc. The exact compositions and concentrations of these inorganic elements that are optimum for a particular plant species can be highly variable. However, prepackaged formulations of these salts that can even include the carbon source, vitamins, hormones, and pH buffers are commercially available.

Secondary Metabolic Requirements A difference between the growth and secondary metabolic phase is that the latter gains importance when approaching the reproductive stages. For example, many of the pigments of flowers are secondary metabolites (e.g., shikonin). Secondary mechanisms are typical responses to stress, such as change in pH (e.g., alkaloid production in Hyocyamus muticus cell cultures is optimum at pH 3.5, while growth is best at 5.0). Similarly, carbon-source concentrations affect Morinda citrifolia cell cultures that grow best at 5 percent sucrose but produce the anthraquinone secondary metabolites optimally at 7 percent. Temperature changes can cause flowering; several plants require a cold treatment to induce flowering. This is called vernalization.

Secondary metabolic pathways in plant cell and tissue cultures seem to be highly controlled by the hormone level in the medium. Another method of eliciting secondary metabolites employs the natural defense mechanisms of the plants that have developed through evolution. For example, gossypol produced by Gossypium hirsutum (cotton) cells is a natural response of the plant when subjected to the infections of the wilt-producing fungus Verticillium dahliae.

Energy for Metabolism

Body Metabolic Reaction

Many metabolic reactions, once activated, proceed spontaneously with a net release of energy. Hydrolysis and molecular rearrangements are examples of spontaneous reactions. The hydrolytic splitting of starch to glucose, for instance, results in a net release of energy. But a great many biochemical reactions are not spontaneous and therefore require an energy input. In living systems this requirement is met by coupling an energy-requiring reaction with an energy releasing reaction. If a sufficient amount of energy is produced by a metabolic reaction, it may be used to synthesize a high-energy compound such as adenosine triphosphate (ATP). When the terminal phosphate linkage is broken, adenosine diphosphate (ADP) and inorganic phosphate are formed, and energy is provided. When sufficient energy becomes available, ATP is reformed from ADP.

In biological systems, the most frequent mechanism of oxidation is the removal of hydrogen, and conversely, the addition of hydrogen is the common method of reduction. Nicotinamide-adenine dinucleotide (NAD) and nicotinamide-adenine dinucleotide phosphate (NADP) are two coenzymes that assist in oxidation and reduction.

These cofactors can shuttle between biochemical reactions so that one drives another, or their oxidation can be coupled to the formation of ATP. However, stepwise release or consumption of energy requires driving forces and losses at each step such that overall efficiency suffers.

Overall redox potential of a system determines the amount of energy that cells can derive from their nutrients. When oxygen is present to be the ultimate acceptor of electrons, complete oxidation of organic molecules yields maximum energy and usually results in the production of H2O and CO2. However, inside animals, in polluted waters, in the benthos (bottom region) of natural waters, and elsewhere, there is little or no free oxygen. In these environments, organisms develop that can partially oxidize substrates or can derive a small amount of energy from reactions where some products are oxidized while others are reduced. The pathways for complete oxidation may be absent and the presence of oxygen can disrupt the mechanisms for anaerobic metabolism so that the cell is quickly killed. The differences in efficiency are striking: Aerobic metabolism of one molecule of glucose can generate bond energy as much as 33 molecules of ATP, while anaerobic metabolism can yield as little as two molecules of ATP.

Natural anaerobic processes accumulate compounds such as ethanol, acetoin, acetone, butanol, lactate, and malate. Products of natural aerobic metabolism are water and carbon dioxide, cell mass, and secondary metabolic products such as antibiotics.

Photosynthesis All living cells synthesize ATP, but only green plants and a few photosynthetic (or phototrophic) microorganisms can drive biochemical reactions to form ATP with radiant energy through the process of photosynthesis. All photosynthetic organisms contain one or more of the group of green pigments called chlorophylls. In plants, these are contained in organelles called chloroplasts. The number per cell of membrane-surrounded chloroplasts varies with species and environmental conditions. In higher plants, numerous chloroplasts are found in each cell of the mesophyll tissue of leaves, while an algal cell may contain a single chloroplast. A chloroplast has a sand-witch of many layers alternating between pigments and enzymatic proteins such that electromagnetic excitation from light becomes chemical bond energy. Prokaryotic organisms have a unique type of chlorophyll and do not possess chloroplasts organelles. Instead, their photosynthetic systems are associated with the cell membrane or with lamellar structures located in organelles known as chromatophores.

Chromatophores, unlike chloroplasts, are not surrounded by a membrane. The net result of photosynthesis is reduction of carbon dioxide to form carbohydrates. A key intermediate is phosphoglyceric acid, from which various simple sugars are produced and disproportionated to form other carbohydrates.

Mutation and Genetic Engineering Exposing organisms to agents such as mustard chemicals, ultraviolet light, and x-rays increases mutation rate by damaging chromosomes. In strain development through mutagenesis, the idea is to limit the mutagen exposure to kill about 99 percent of the organisms. The few survivors of this intense treatment are usually mutants. Most of the mutations are harmful to the cell, but a very small number may have economic importance in that impaired cellular control may result in better yields of product. The key is to have a procedure for selecting out the useful mutants. Screening of many strains to find the very few worthy of further study is tedious and expensive. Such screening that was so very important to biotechnology a few decades ago is becoming obsolete because of genetic improvements based on recombinant DNA technology.

Whereas mutagenic agents delete or scramble genes, recombinant DNA techniques add desirable genetic material from very different cells. The genes may come from plant, animal, or microbial cells, or in a few instances they may be synthesized in the laboratory from known nucleic acid sequences in natural genes. Opening a chromosome and splicing in foreign DNA is simple in concept, but there are complications.

Genes in fragments of DNA must have control signals from other nucleic acid sequences in order to function. Both the gene and its controls must be spliced into the chromosomes of the receiving culture.

Bacterial chromosomes (circular DNA molecules) are cut open with enzymes, mixed with the new fragments to be incorporated, and closed enzymatically. The organism will acquire new traits. This technique is referred to as recombinant technology.

There are many tricks and some art in genetic engineering. Examples would be using bacteriophage infection to introduce a gene for producing a new enzyme in a cell. Certain strains of E. coli, B. subtilis, yeast, and streptomyces are the usual working organisms (cloning vectors) to which genes are added. The reason for this is that the genetics of these organisms is well understood and the methodology has become fairly routine.

Cell and Tissue Cultures

Cell Production and Tissue Cultures

Mammalian Cells Unlike microbial cells, mammalian cells do not continue to reproduce forever. Cancerous cells have lost this natural timing that leads to death after a few dozen generations and continue to multiply indefinitely. Hybridoma cells from the fusion of two mammalian lymphoid cells, one cancerous and the other normal, are important for mammalian cell culture. They produce monoclonal antibodies for research, for affinity methods for biological separations, and for analyses used in the diagnosis and treatment of some diseases.

However, the frequency of fusion is low. If the unfused cells are not killed, the myelomas will overgrow the hybrid cells. The myelomas can be isolated when there is a defect in their production of enzymes involved in nucleotide synthesis. Mammalian cells can produce the necessary enzymes and thus so can the fused cells. When the cells are placed in a medium in which the enzymes are necessary for survival, the myelomas will not survive. The unfused normal cells will die because of their limited life span. Thus, after a period of time, the hybridomas will be the only cells left alive.

A hybridoma can live indefinitely in a growth medium that includes salts, glucose, glutamine, certain amino acids, and bovine serum that provides essential components that have not been identified. Serum is expensive, and its cost largely determines the economic feasibility of a particular culture system. Only recently have substitutes or partial replacements for serum been found. Antibiotics are often included to prevent infection of the culture. The pH, temperature and dissolved oxygen, and carbon dioxide concentration must be closely controlled.

The salt determines the osmotic pressure to preserve the integrity of the fragile cell. Most glucose is metabolized to lactate because glycolysis is usually much faster than uptake rate of glycolytic intermediates. Glutamine acts as the primary source of nitrogen as well as providing additional carbon and energy. After glutamine is partially oxidized to glutamate, it can enter the TCA cycle and emerge as pyruvate. It has been estimated that between 30 and 65 percent of the cell energy requirement is derived from glutamine metabolism when both glucose and glutamine are available. Ammonia is produced in the deamination of glutamine to form glutamate and in the formation of alpha-ketoglutarate.

Plant Cells and Tissues It is estimated that today some 75 percent of all pharmaceuticals originate in plants. Typically, these compounds are derived from the secondary metabolic pathways of the cells. When plant or animal cells are cultured, concepts from microbiology come into play. Only specialized cells are used, and these can be improved with mutation, selection, and recombinant DNA techniques.

One very major difference between cell and tissue cultures and most microbiological processes is very high susceptibility to contamination by foreign organisms. Most microorganisms grow rapidly and compete well; some are aided by their own changes to the environment.

When a microbial process changes the pH to be far from neutrality or when the product such as ethanol is inhibitory to other organisms, growth of contaminants is discouraged. Cell and tissue cultures require rich media and are characterized by slow growth rates.

There is seldom any protection by the products of the process. Optimum conditions for production of the secondary metabolites are not likely to be the same as for growth. Economics may hinge on a good balance of growing sufficient cells and favoring product formation.

Only a few biochemicals derived from plant cell and tissue cultures have high volume/low value products, but some have sizeable markets as specialty chemicals such as dyes, fragrances, insecticides, and pesticides. These differ from the low volume/very high value compounds that typify life-saving drugs and pharmaceuticals. Examples for both of these categories are listed in Table 1 along with the plant species of origin.

Because of cell specialization, some products are produced in cultures of those cellular types. Three main classifications of the types of plant cell and tissue cultures are:

Undifferentiated cell cultures. Aggregate clumps of cells on solid media (callus) or in liquid media (suspension) Protoplast cultures. Cellular tissues devoid of cell wall material in culture Organ cultures. Differentiated tissues of shoots, roots, anthers, ovaries, or other plant organs in culture.