Plant Biotechnology

S. Umesha

• Publisher: CRC Press
• Year: 2019
• ISBN:
  9780429614620
• Edition: 1
• Topic: Sciences biologiques
• Subtopic: Botanique

Plant Biotechnology comprehensively covers different aspects of the subject based on the latest outcomes of this field. Topics such as tissue culture, nutrient medium, micronutrients, macronutrients, solidifying agents/supporting systems, and growth regulators have been dealt with extensively. The book also discusses in detail plant genetic engineering for productivity and performance, resistance to herbicides, insect resistance, resistance to abiotic stresses, molecular marker aided breeding, molecular markers, types of markers, and biochemical markers. Different aspects of important issues in plant biotechnology, commercial status and public acceptance, biosafety guidelines, gene flow and IPR have been also thoroughly examined. This book caters to the needs of graduate, postgraduate and researchers.

Please note: This volume is Co-published with The Energy and Resources Institute Press, New Delhi. Taylor & Francis does not sell or distribute the Hardback in India, Pakistan, Nepal, Bhutan, Bangladesh and Sri Lanka

Tissue Culture

1

1.1 INTRODUCTION TO CELL AND TISSUE CULTURE

Totipotency is one of the most wonderful phenomena of plant cells. An entire organism can be produced by this inherent capacity of cells when a suitable nutrient medium is present. In plant tissue culture, plant cells, tissues, or segments are grown for generating large numbers of new cells, tissues, or plants. Plant tissue culture is also used to study development of plant cells, and it provides mechanisms of genetic engineering and produces valuable chemicals found in plant cells. For understanding many applied aspects of plant science, it is essential to study the techniques of plant tissue culture. These techniques have been used in the past to study totipotency and determine the roles of hormones in cytodifferentiation and organogenesis. At present, an insight into plant molecular biology and gene regulation can be obtained from tissue culture and genetic engineering of plants. Plant tissue culture techniques also play a central role in innovative areas of applied plant science, such as plant biotechnology and agriculture.

Cloning is a very familiar concept for people who study the reproducibility of plants. Cloning is the process used to produce exact and multiple copies of a parent plant. Cuttings and grafts can be used for cloning. Cloning can also occur naturally through agents such as the propagules dispersed by spider plants. Much smaller pieces of tissue are used by plant tissue culture in the cloning process. Plant tissue culturists have many possibilities in this field and can do more than simply cloning plants. They can place new plants into culture or introduce new methods of working with plant cultures.

At the beginning of an experiment, a plant tissue culturist should make purposeful ideas about its necessity. There are different types of in vitro tissue culture approaches. The term in vitro refers to growing pieces of plants separately in artificial conditions. For instance, plant products can be harvested from select plants cultured or cloned as suspended cells. Moreover, tissue culture procedures are required for the management of genetically modified cells used to grow transgenic whole plants. These methods are also necessary for forming somatic haploid embryos to generate homozygous plants. Hence, tissue culture techniques will remain prominent in academic and applied plant sciences.

1.2 TISSUE CULTURE MEDIA (COMPOSITION AND PREPARATION)

The composition of the culture medium is an important factor that governs the growth and morphogenesis of plant tissues in the culture. Cultured plant cells have the same basic nutrient requirements as the whole plant. Some or all of the following components are present in the culture media of plant tissues and cells: micronutrients, macronutrients, amino acids or other nitrogen supplements, vitamins, sugar, solidifying agents or support systems, other undefined organic supplements, and growth regulators. Common media formulations used in majority of cell and tissue culture work include those described by White (1963); Murashige and Skoog (1962); Gamborg, Miller, and Ojima (1968); Schenk and Hildebrandt (1972); Nitsch and Nitsch (1969); and Lloyd and McCown (1980). The media high in macronutrients include the Murashige and Skoog medium, Schenk and Hildebrandt medium, and Gamborg B5 medium.

1.2.1 Macronutrients

Macronutrients consist of six major elements that are required for the growth of plant tissue. They include nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), and sulphur (S). The optimum concentration of each nutrient required for maximum growth rate depends on the species.

For proper plant cell growth, the culture media must contain at least 25–60 mM of inorganic nitrogen. Although plant cells may be grown on nitrates alone, better results can be obtained if nitrate and ammonium are used as nitrogen sources. For their cell growth, some species require ammonium or another source of reduced nitrogen. Nitrates are usually supplied in the range of 20–25 mM, while ammonium concentrations typically lie in the range 2–20 mM. However, ammonium concentrations in excess of 8 mM may be deleterious to cell growth in certain species. Cells can also be grown on a medium having ammonium as the only source of nitrogen if one or more of the tricarboxylic acid (TCA) cycles (e.g., citrate, succinate, or malate) are included in the medium at approximately 10 mM concentrations. When nitrate and ammonium are used together in the culture medium as source of nitrogen, the ammonium ions are consumed more rapidly and before the nitrate ions.

Potassium is an important element for cell growth in most plants. It is present in the form of nitrates or chlorides in most media at concentrations of 20–30 mM. When all other requirements for cell growth are met, the optimum concentrations of phosphorous, magnesium, sulphur, and calcium range from 1 to 3 mM. These nutrients may be required in higher concentrations when other nutrients are deficient.

1.2.2 Micronutrients

Iron (Fe), manganese (Mn), zinc (Zn), boron (B), copper (Cu), and molybdenum (Mo) constitute the micronutrients essential for plant cell and tissue growth. Culture media are mostly prepared from the chelated forms of iron and zinc. Of all the micronutrients, iron is the most essential. Iron citrate and tartrate may be used in the culture media. But these compounds sparsely dissolve and are mostly precipitated after the media are formed. To resolve this problem, Murashige and Skoog (1962) used an ethylenediaminetetraacetic acid (EDTA)–iron chelate. Cobalt (Co) and iodine (I) may be added in certain media, but it is not established whether they can promote cell growth. Although sodium (Na) and chlorine (Cl) are not essential for cell growth, they are often added in some media. Usually copper and cobalt, iron and molybdenum, iodine, zinc, manganese, and boron are added to culture media at the concentrations of 0.1, 1, 5, 5–30, 20–90, and 25–100 μM, respectively.

1.2.3 Carbohydrates

Scientists like to use sucrose as the carbohydrate in the plant cell culture media. In some cases, instead of sucrose, glucose or fructose can also be used. While glucose is as effective as sucrose, fructose is not so effective. Some other carbohydrates have also been tested, such as starch, lactose, rafinose, maltose, and galactose. The concentration of sucrose in the culture media generally ranges between 2% and 3%. Autoclaved fructose can be dangerous for cell growth.

The culture medium should be supplied with carbohydrates because only a few isolated plants cell lines are completely autotrophic, that is, capable of meeting their own carbohydrate needs by assimilating carbon dioxide during photosynthesis.

1.2.4 Vitamins

The vitamins required for development and growth are normally produced in plants. Vitamins catalyse various metabolic processes in plants. In plant cells and tissues grown in vitro, some vitamins may behave as limiting factors for cell growth. Vitamins frequently used in cell and tissue culture media are thiamine (B1), nicotinic acid, pyridoxine (B6), and myo-inositol. All cells normally require thiamine at concentrations of 0.1–10 mg/L for growth. Although nicotinic acid and pyridoxine are often added to the culture media, they are not necessary for cell growth in many species. Usually nicotinic acid and pyridoxine are used at the concentrations of 0.1–5.0 mg/L and 0.1–10 mg/L, respectively. Many vitamin stock solutions contain myo-inositol. Even though myo-inositol is a carbohydrate and not a vitamin, it is reported to stimulate growth in certain cell cultures. Its presence is not essential in the culture medium. However, it stimulates cell growth in most species when it is present in small quantities. Myo-inositol is mostly used in cell and tissue culture media at concentrations in the range 50–5000 mg/L.

Some of the cell culture media also contain other vitamins, such as ascorbic acid, biotin, folic acid, p-aminobenzoic acid, pantothenic acid, riboflavin, and vitamin E (tocopherol). Required in negligible amounts in plant cell cultures, these vitamins are generally not regarded as growth-limiting factors. The culture medium generally requires these vitamins only when the thiamine concentration is below the desired level or when it is preferable to cultivate cells at very low population density.

1.2.5 Amino Acids or other Nitrogen Supplements

Although cultured cells are normally capable of synthesizing all the required amino acids, certain amino acids or amino acid mixtures may be added to stimulate further cell growth. It is particularly important to use amino acids for forming cell cultures and protoplast cultures. The nitrogen present in amino acids is immediately available to plant cells, and it can be absorbed more rapidly than inorganic nitrogen.

Some of the common sources of organic nitrogen used in the culture media include amino acid mixtures (e.g., casein hydrolysate), adenine, L-asparagine, and L-glutamine. Generally, casein hydrolysate is used in the concentration range 0.05%–0.1%. Care must be taken when amino acids are added alone because they may hinder cell growth. The amino acids commonly added in the culture media to augment cell growth include glycine at 2 mg/L, glutamine up to 2 mg/L, asparagine at 100 mg/L, L-arginine and cysteine at 10 mg/L, and L-tyrosine at 100 mg/L. Morphogenesis in cell cultures is stimulated by the addition of tyrosine, but it should be used only in an agar medium. On supplementing the culture medium with adenine sulphate, the cell growth and shoot formation can be greatly enhanced.

1.2.6 Undefined Organic Supplements

When a wide variety of organic extracts are added to culture media, favourable tissue responses are obtained. Some tested supplements include coconut milk, ground banana, malt extracts, orange juice, protein hydrolysates, tomato juice, and yeast extracts. However, the use of undefined organic supplements should be the last resort, and only coconut milk and protein hydrolysates are used to any extent today. The concentration of protein (casein) hydrolysates generally added to the culture media is 0.05%–0.1%, while that of coconut milk is 5%–20% (v/v).

Activated charcoal (AC) may have beneficial effects on the culture media. These effects can be because of one of the following factors: absorption of growth regulators from the culture medium, absorption of inhibitory compounds, and darkening of the medium. Growth is inhibited in the presence of AC mostly because phytohormones get absorbed by AC. 1-Naphthaleneacetic acid (NAA), 6-benzylaminopurine (BAP), 6-γ-γ-dimethylallylaminopurine, indole-3-acetic acid (IAA), and kinetin all bind to AC. Cell growth is stimulated by AC because it binds to toxic phenolic compounds produced during culture. Before being added to the culture medium at the concentration of 0.5%–3.0%, AC is mostly acid washed.

1.2.7 Solidifying Agents or Support Systems

The most common gelling agent used for preparing semisolid and solid tissue culture media is agar. There are several advantages of using agar over other gelling agents. It can easily melt and solidify. When agar is mixed with water, it forms a gel that melts at approximately 60–100°C and solidifies at approximately 45°C. This makes agar gels stable at all feasible incubation temperatures. Besides, constituents of the media do not react with agar gels and plant enzymes do not digest them. The firmness of agar gel depends on the concentration and brand of agar used and the pH of the culture medium. The concentrations of agar commonly used in culture media lie in the range 0.5%–1%. At this range, agar forms a firm gel at various pH values typical of plant cell culture media.

Gelrite is another gelling agent commonly used for research and commercial purposes. To form a clear gel, this synthetic product must be used at a concentration of 1.25–2.5 g/L, which aids in detecting contamination. There are alternative methods of support systems such as filter paper bridges, filter paper wicks, perforated cellophane, polyester fleece, and polyurethane foam. Whether agar or other supporting agents will be more suitable for the growth of explants depends on plant species.

1.2.8 Growth Regulators

In plant tissue culture, there are four important classes of growth regulators: (i) auxin, (ii) cytokinin (CK), (iii) gibberellin (GA3), (iv) and abscisic acid (ABA). Skoog and Miller (1957) reported that the type and extent of organogenesis in plant cell cultures depend on the ratio of auxin to CK. Usually, auxin and CK are added to the culture media to promote morphogenesis. However, the ratio of hormones required for root and shoot induction is not the same universally. There exists considerable variability among genera, species, and even cultivars regarding the amount and type of auxin and CK required for morphogenesis.

Auxins commonly used in plant tissue culture media include 1H-indole-3-acetic acid (IAA), 1H-indole-3-butyric acid (IBA), 2,4-dichlorophenoxyacetic acid (2,4-D), and NAA. The only naturally occurring au...