Plant Tissue Culture Technology: Methods and Applications
Introduction to Plant Tissue Culture (PTC)
Plant tissue culture (PTC), developed around the 1950s, was a significant addition to plant breeding methods. Conventional breeding techniques often struggled to meet the required demand for crops, making tissue culture a major advancement in breeding practices.
PTC is defined as the in vitro aseptic culture of cells, tissues, or whole plants under controlled nutritional and environmental conditions, primarily used to produce clones of plants.
The Principle of Totipotency
The technique relies fundamentally on the property of totipotency of plant cells. Totipotency means that any cell from any part of the plant can be used to generate a whole new plant. It is the capacity of a single cell to express the entire genome during cell division. The ability of cells to change their metabolism, growth, and development is essential for the regeneration of the entire plant.
PTC utilizes parts of a plant (explants) to generate multiple, genetically identical copies of the parent plant in a very short duration.
Types of Plant Tissue Culture
- Seed Culture
- Embryo Culture
- Callus Culture
- Organ Culture
- Protoplast Culture
- Anther Culture
Procedure of Plant Tissue Culture
The part(s) of the plant used for culturing is known as explants. The explants are cultured in vitro on a nutrient medium that is formulated to fulfill their nutritional requirements. The nutrient medium must provide the following essential components:
Essential Components of the Nutrient Medium
Macronutrients
This includes elements like nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), and sulfur (S), which are required for proper growth and morphogenesis.
Micronutrients
Elements like iron (Fe), manganese (Mn), zinc (Zn), etc., which are also crucial for tissue growth.
Carbon or Energy Source
This is one of the most crucial ingredients. Sucrose is the most widely used carbon source among other carbohydrates that serve to provide C, H, and O.
Vitamins, amino acids, and other inorganic salts.
Role of Phytohormones and Callus Formation
Apart from these basic components, the culture medium also supplies phytohormones (plant growth regulators) to the tissues, which facilitate their morphogenesis as required. The explant tissues first lose their specificity to form a hard lump known as callus. The callus then differentiates to develop a plant organ or a whole new plant, depending upon the quantity and composition of the supplied phytohormones.
The entire process requires strict aseptic conditions to be maintained at all times, as a single contamination can ruin an entire batch of plants.
Applications of Plant Tissue Culture
Micropropagation and Somaclones
Tissue culture is primarily used for micropropagation—the process of developing thousands of genetically identical plants (known as somaclones) from a single parent plant.
The method offers a significant advantage as it can be used to develop disease-free plants from diseased parent plants by using their meristems (apical and axillary) as explants.
Since PTC produces new plantlets in massive quantities, it is used extensively for the commercial production of important plants, including food crops like tomato, banana, and apple. A notable example is the exponential growth observed in orchid farming, facilitated by the availability of millions of plantlets through tissue culture methods.
Plant tissue culture technology is now essential for plant propagation, disease eradication, and the generation of secondary metabolites, in addition to its use in research.
Advantages of Plant Tissue Culture
- Facilitates the study and preservation of plant cells, including totipotency, nutrition, metabolism, division, and differentiation.
- Enables morphogenesis and plant regeneration from individual cells or tissues through organogenesis or somatic embryogenesis.
- Allows for the generation of variations through in vitro culture.
- Supports the evolution of haploids through anther, pollen, and ovule culture.
- Crucial for wide hybridization programs through ovule, ovary, and embryo cultures, helping to overcome both pre-zygotic and post-zygotic sterility mechanisms.
- Efficient micropropagation of plant materials.
- Enables in vitro selection of mutants tolerant to biotic and abiotic stresses.
- Used for in vitro culture and secondary metabolite biosynthesis.
- Essential for plant genetic engineering using DNA transfer and in vitro culture techniques.
Optimizing Plant Tissue Culture Conditions
The choice of medium is based on the specific plant species and the explants used for culture to ensure an optimal response. The plant tissue culture media must contain all the nutrients required for a plant’s proper growth and development.
The composition typically includes macronutrients, micronutrients, vitamins, other organic ingredients, plant growth regulators, a carbon source, and, for solid media, gelling agents. Culture variables like temperature, pH, light intensity, and humidity also play an important role in the success of tissue culture.
Key Medium Components Detailed
Minerals
The minerals consist of macronutrients (such as nitrogen, potassium, phosphorus, calcium, magnesium, and sulfur) and micronutrients (such as iron, manganese, zinc, boron, copper, molybdenum, and cobalt).
Vitamins
Vitamins are necessary for the healthy growth of plant cultures. Commonly used vitamins include thiamine (vitamin B1), pyridoxine (B6), and nicotinic acid (niacin). Other vitamins such as biotin, folic acid, ascorbic acid (vitamin C), and vitamin E (tocopherol) are sometimes added.
Carbon Source
Plants require an external carbon source, typically sugar. The most commonly used carbon source is sucrose. Other sources include glucose, maltose, and sorbitol.
pH Level
The pH of the culture medium is vital as it influences the uptake of various components and regulates a wide range of biochemical reactions. Most media are adjusted to a pH of 5.2–5.8, though a higher pH may be required for certain cultures.
Historical Milestones in Tissue Culture
The concept of growing cells outside an original host body, provided stringent external sterile conditions were created, was first presented by Theodor Schwann in 1832. This seemingly improbable statement was proven by Wilhelm Roux in 1835, who successfully cultured embryonic chicken cells using a salt solution medium.
In 1839, Reichinger proposed the first parameter for this method, suggesting that fragments required a minimum thickness of 1.5mm to grow successfully.
Later, in 1907, American zoologist Ross Granville Harrison successfully cultured nerve cells from a frog in solidified lymph. Due to his contributions, Harrison is often titled the Father of Tissue Culture.
In 1929, the first organ culture was successfully performed in England by D.H. Fell.
The Dawn of Plant Tissue Culture
Although general tissue culture began in the 19th century, plant tissue culture only started developing around 1898. Plant tissue culture has since made significant waves in the plant environment due to the potential for increased yield, healthier genes, and sterile propagation.
Gottlieb Haberlandt, a German Botanist, made the first attempt to use the in vitro method to grow plant tissues in 1898 and 1902. He tried to cultivate individual palisade cells from leaves in Knop’s salt solution supplemented with sucrose. The cells he used were varied, coming from:
- The leaves (palisade tissues)
- The piths
- The epidermis and epidermal hairs
In his 1902 experiment, the cells sustained for a month and stored starch but ultimately did not divide. Despite this failure, Haberlandt is considered the Father of Plant Tissue Culture because his experiment set the stage for developing the technology.
Further milestones include:
- 1922: The first root tips were cultured and maintained for 20 weeks using subculturing techniques.
- 1930s: It was recognized that B-vitamins and auxin (IAA) were key components in growing root cultures.
- 1934: French scientist Roger J. Gautheret achieved encouraging results with culturing cambial tissues of carrots.
Over the following century, researchers continued to identify the most important parameters guiding modern tissue culture processes.
Whether used for commercial profit or personal pleasure, the tissue culture method remains a powerful tool for plant propagation.