What is Plant Tissue Culture?
Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues, or organs under sterile conditions on a nutrient culture medium of known composition. It is widely used to produce clones of a plant in a method known as micropropagation.
Different techniques in plant tissue culture may offer certain advantages over traditional methods of propagation, including:
- The production of exact copies of plants that produce particularly good flowers, fruits, or other desirable traits.
- To quickly produce mature plants.
- To produce a large number of plants in a reduced space.
- The production of multiples of plants in the absence of seeds or necessary pollinators to produce seeds.
- The regeneration of whole plants from plant cells that have been genetically modified.
- The production of plants in sterile containers allows them to be moved with greatly reduced chances of transmitting diseases, pests, and pathogens.
- The production of plants from seeds that otherwise have very low chances of germinating and growing, i.e. orchids and Nepenthes.
- To clean particular plants of viral and other infections and to quickly multiply these plants as ‘cleaned stock’ for horticulture and agriculture.
- Reproduce recalcitrant plants required for land restoration
- Storage of genetic plant material to safeguard native plant species.
Plant tissue culture relies on the fact that many plant parts have the ability to regenerate into a whole plant (cells of those regenerative plant parts are called totipotent cells which can differentiate into various specialized cells).
Single cells, plant cells without cell walls (protoplasts), pieces of leaves, stems or roots can often be used to generate a new plant on culture media given the required nutrients and plant hormones.
History of Plant Tissue Culture
Gottlieb Haberlandt, now known as the father of plant tissue culture, proposed the theoretical basis of plant tissue culture in 1902. He experimented with photosynthetic leaf cells but could not induce any growth.
However, he predicted that one can obtain artificial embryos from vegetative cells using this culturing technique and established the concept of totipotency.
Types of Plant tissue culture
- Seed Culture
- Embryo Culture
- Callus Culture
- Organ Culture
- Protoplast Culture
- Anther Culture
#1. Seed Culture.
Seed culture is a type of tissue culture that is extensively employed in the cultivation of orchids and other plants. Plant tissues are extracted from an in-vitro grown plant and put in an artificial environment where they may develop for this process.
If plant material is directly used in this method, it must be sanitised to minimise tissue injury and enhance effective regeneration.
When obtaining explants from in vitro-derived plants and propagating orchids, culture seeds are a must. Because explant sources might cause tissue injury and interfere with regeneration, sterile procedures are essential for plant materials that will be used directly.
Culture seeds to create sterile seedlings is the best solution in that case. It takes a long time to clone orchids in vivo.
#2. Embryo Culture.
Embryo culture involves isolating and growing an immature or mature zygotic embryo under sterile conditions on an aseptic nutrient medium with the goal of obtaining a viable plant.
Embryo culture is one of the extensively and commonly used techniques for embryo rescue. In this method, seeds are collected from controlled pollination plants before an embryo is expected to abort.
After this, the embryo is isolated and excised in a sterile condition, followed by directly culturing it on the culture medium to obtain viable plants.
Based on the type of embryo introduced into the culture medium, the embryo culture is of two types:
1. Mature Embryo Culture: It’s the process of culturing mature and ripped embryos in suitable laboratory conditions. It’s done when the embryos don’t survive in natural in vivo conditions, become dormant for a long time, or when seed germination is inhibited.
2. Immature Embryo Culture: It’s used to culture immature, weak, or small embryos that fail to develop into hybrid plants due to endosperm degeneration in natural conditions. This is also known as embryo rescue, as by culturing the embryos they are being rescued and promoted to develop into a complete plant.
#3. Callus Culture.
Callus culture is a technique where a piece of plant tissue (explant) is grown in a nutrient medium under controlled conditions. This stimulates the explant to form a mass of undifferentiated cells called callus. Much like a plant stem cell, this callus has the potential to develop into a whole plant through plant regeneration.
However, callus cultures display significant heterogeneity. Researchers have classified them into distinct subgroups based on their macroscopic characteristics (observable without magnification).
For example, callus lacking signs of organ regeneration is typically categorized as friable or compact. Conversely, some callus exhibits varying degrees of organ formation, categorized as rooty callus, shooty callus, or embryonic callus depending on the organs they produce.
Notably, studies in the model plant Arabidopsis thaliana have revealed distinct gene expression profiles among different callus types This highlights the inherent diversity within the callus category, encompassing cells with varying degrees of differentiation potential.
#4. Organ culture.
As the name suggests, organ culture is in vitro generation of plants using plant organs such as the leaf, node, internode, shoot, root, axillary bud, and seedling.
Organ culture can be defined as the organs or plant parts culturing in an artificial media or a culture from isolated medium. Any part of plant can serve as explants in organ culture-like shoot (for shoot tip culture), root (for root tip culture), leaf (for leaf culture), and flower.
If you aim to avoid somaclonal variation in your experiments, organ culture is the preferable technique. Some other applications of the technique include the production of secondary metabolites; the study of the pattern of growth, differentiation, and development of organisms; and biochemical and molecular functions of an organ or tissue.
The best advantage of organ culture is the maintenance of organ structure and function even after culturing. It preserves the originality of the plant and shows the same characteristics as it would show in vivo.
#5. Protoplast Culture.
Protoplast culture refers to the process in which whole plants are developed from the culture of cells without cell wall. This technique was discovered over 10 decades ago and is still widely used in plant breeding and crop improvement programs.
Protoplasts are of great interest because they are totipotent. It means that when cultured, they can grow, divide, and differentiate into a whole plant.
They are sensitive, versatile and any type of plant, organ, and tissue can be used to produce them. Protoplasts are usually derived from leaf tissues, but callus, cell suspension, and pollen grains are also commonly used.
The first isolation of plant protoplasts was achieved in 1892 by Klercker in onion. Today, there is a protocol for protoplast production for nearly all crops.
#6. Anther Culture.
Anther culture is a doubled haploid technique for producing homozygous lines. In coconut, a tree species reported to be recalcitrant for tissue culture, a successful doubled haploid protocol was established through anther culture. All the factors affecting androgenesis induction have been optimized.
Anther culture is a type of tissue culture technique used to produce haploids and dihaploids. It is simpler than the pollen culture technique. It uses microspores or anthers for plant regeneration.
The first successful report of anther culture was published in the 1970s by Guha and Maheshwari on pollen grains of Datura. Since then it has been used in numerous species (around , mainly rice (O. sativa) and tobacco (N. tabacum).
During the process, the anthers are excised at a critical stage from an unopened flower bud aseptically. Then, they are cultured on a nutrient medium for the formation of callus tissue or embryoids that give rise to haploid plantlets through embryogenesis or organogenesis.
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 caters to fulfil its nutritional requirements. The nutrient medium must provide the following:-
- Macronutrients – This includes elements like nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), sulfur (S) which is required for proper growth and morphogenesis.
- Micronutrients – Elements like iron (Fe), manganese (Mn), zinc (Zn) etc., which are also crucial to the growth of tissues.
- Carbon or Energy source – This is one of the most crucial ingredients of the nutrient media. 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.
Apart from these, the culture media also serves as a medium for supplying phytohormones or plant growth regulators to the issues which bring about their morphogenesis as per requirement. The tissues of the explants first lose their specificity to form a hard brown lump known as callus.
The callus then splits to develop a plant organ or a whole new plant depending upon the quantity and composition of phytohormones supplied. The entire process requires strict aseptic conditions to be maintained at all times as a single contamination can ruin an entire batch of plants.
Uses of Plant tissue culture
Plant tissue culture is used widely in the plant sciences, forestry, and horticulture. Applications include:
- The commercial production of plants used as potting, landscape, and florist subjects, which uses meristem and shoot culture to produce large numbers of identical individuals.
- To conserve rare or endangered plant species.
- A plant breeder may use tissue culture to screen cells rather than plants for advantageous characters, e.g. herbicide resistance/tolerance.
- Large-scale growth of plant cells in liquid culture in bioreactors for production of valuable compounds, like plant-derived secondary metabolites and recombinant proteins used as biopharmaceuticals.
- To cross distantly related species by protoplast fusion and regeneration of the novel hybrid.
- To rapidly study the molecular basis for physiological, biochemical, and reproductive mechanisms in plants, for example in vitro selection for stress-tolerant plants.
- To cross-pollinate distantly related species and then tissue culture the resulting embryo which would otherwise normally die (Embryo Rescue).
- For chromosome doubling and induction of polyploidy, for example doubled haploids, tetraploids, and other forms of polyploids. This is usually achieved by the application of antimitotic agents such as colchicine or oryzalin.
- As a tissue for transformation, followed by either short-term testing of genetic constructs or regeneration of transgenic plants.
- Certain techniques such as meristem tip culture can be used to produce clean plant material from virused stock, such as sugarcane, potatoes and many species of soft fruit.
- Production of identical sterile hybrid species can be obtained.
- Large scale production of artificial seeds through somatic embryogenesis.