Tissue Culture | Botany Optional for UPSC PDF Download

What are the different types of plant tissue culture?

Following are types of plant tissue culture:

1. Cell or suspension culture
2. Explant culture
3. Callus culture
4. Protoplast culture
5. Embryo culture
6. Anther and pollen culture
7. Ovule culture
8. Ovary culture

Cell or suspension culture

• Cell or suspension culture can be initiated directly from explants (plant tissue) or from callus (undifferentiated plant tissue).
• Pieces of undifferentiated callus are transferred to a liquid medium and continuously agitated to create a suspension culture.
• Suspension culture involves the growth of single cells and cell clumps in the liquid medium.

Single Cell Culture Methods

• Filter Paper Raft Nurse Tissue Technique:
  • Single cells are placed on filter paper pieces (8x8mm) that are situated on established callus cultures.
  • Single cells receive nutrients from the callus exudates diffusing through the filter papers.
• Microchamber Technique:
  • A microchamber is created using a microscopic slide and coverslip or a cavity slide.
  • Single cells are suspended in a conditioned medium, and a drop is placed onto a coverslip, which is then inverted into the slide cavity.
  • This method allows for microscopic observation and easy sub-culturing on a petri dish.
• Microdrop Method:
  • A specially designed dish with smaller outer chambers filled with distilled water to prevent cell desiccation and larger chambers containing numerous micron-cells is used.
  • Microdrops of culture medium (0.25-0.5 ml) are distributed in the micron-cells, with an average of one cell per droplet.
  • The dish is sealed with parafilm.
• Bergmann’s Plating Technique:
  • Free cells are suspended in a liquid medium, and if cell aggregates are present, they are filtered.
  • The culture medium is cooled and maintained at 35°C in a water bath.
  • Cells are mixed with the medium and poured into a petri dish, allowed to solidify, and sealed with parafilm.
  • Colonies that develop are isolated and cultured separately.

Applications of single cell

Single cell cultures have several important applications in plant tissue culture and biotechnology:

• Induction of Somatic Embryos and Shoots: Single cells can be used as starting materials for the induction of somatic embryos and shoots. This is valuable in plant propagation and regeneration, allowing for the production of multiple plants from a single cell.
• In Vitro Mutagenesis and Selection of Mutants: Single cells can be subjected to mutagenesis techniques to induce genetic mutations. These mutated cells can then be screened for desirable traits, leading to the selection of mutants with specific characteristics, such as disease resistance or improved agronomic traits.
• Genetic Transformation Studies: Single cells are often used in genetic transformation studies to introduce foreign genes (transgenes) into plant cells. This technology is essential for creating genetically modified (GM) plants with desired traits, such as herbicide resistance or enhanced nutritional content.
• Production of Secondary Metabolites: Some plant species produce valuable secondary metabolites, such as alkaloids, flavonoids, and essential oils. Single cell cultures can be used to produce and harvest these secondary metabolites for pharmaceutical, cosmetic, or industrial purposes.

Explant Culture

Explants are essential components in plant tissue culture, and their selection plays a critical role in the success of the culture. Here are key points about explants:

• Definition: An explant is any excised part of a plant that has the potential to develop into a whole plant when placed in a suitable culture medium.
• Presence of Parenchyma Tissues: Explants should ideally contain parenchyma tissues. Parenchyma tissues are versatile and capable of cell division and growth, making them suitable for tissue culture.
• Selection Criteria: Explants should be chosen from young and healthy parts of the plant. These regions are more likely to contain actively dividing cells, which are essential for tissue culture success.
• Types of Explants: Explants can be obtained from various plant parts, including stems, rhizomes, tubers, roots, cotyledons (seed leaves), and hypocotyls (the region between the cotyledons and the root).
• Tissue Development: In tissue culture, explants undergo a series of processes, including cell division, cell elongation, and cell differentiation, leading to the development of new plant tissue.

The selection of an appropriate explant is a critical step in initiating and establishing a successful plant tissue culture. The choice of explant type and its condition greatly influences the culture's success and the subsequent regeneration of whole plants.

Callus Culture

Callus is a critical component of plant tissue culture and has specific characteristics and factors to consider:

• Callus Definition: Callus is an undifferentiated, amorphous mass of loosely arranged, thin-walled parenchyma cells that develop from proliferating cells of the parent tissue.
• Biological Potential: Callus has the biological potential to differentiate into various plant organs, including roots, shoots, and embryos. Ultimately, it can develop into a whole plant through the regeneration process.
• Subculturing: Callus cultures should be subcultured regularly, typically every 28 days. This practice helps prevent nutrient depletion and the accumulation of toxic metabolites, ensuring the long-term viability of the culture.
• Factors Affecting Callus Culture:
  • Environmental Factors: The culture environment, including temperature, humidity, and light conditions, can influence callus growth and development.
  • Nutrient Composition: The concentration and ratio of plant growth regulators, such as auxins and cytokinins, in the culture medium are critical for controlling callus formation and differentiation.
  • Explant Source: The choice of explant source (the tissue used to initiate the culture) can significantly affect callus initiation and characteristics.

Overall, callus plays a central role in plant tissue culture as it serves as the starting point for regenerating whole plants. Proper management of callus cultures and consideration of various factors are essential for successful tissue culture experiments and plant regeneration.

Somatic Embryogenesis

Somatic embryogenesis is a fascinating process in plant tissue culture that involves the development of embryos from somatic or vegetative cells.
Here are some key concepts related to embryogenesis:

• Zygotic Embryo: This is an embryo formed from a zygote, which is the result of fertilization between male and female gametes.
• Non-Zygotic Embryo: Non-zygotic embryos are embryos that originate from parts of the plant other than the zygote. These can include somatic or vegetative cells.
• Somatic Embryo: Somatic embryos are embryos that develop from somatic or vegetative parts of the plant, such as leaf or stem cells, rather than from the zygote.
• Parthenogenic Embryo: A parthenogenic embryo is derived from an unfertilized egg. It develops without fertilization.
• Androgenic Embryo: Androgenic embryos are embryos that develop from pollen. This process is often used in plant breeding to produce haploid plants for further genetic studies.
• Historical Milestone: In 1959, somatic embryogenesis was achieved for the first time from callus culture and suspension culture of carrot (Daucus carota). This breakthrough opened the door to studying and utilizing somatic embryogenesis in plant tissue culture.
• Nutrient Factors: Major nutrient factors that influence somatic embryogenesis include auxins, typically used at high concentrations, and smaller amounts of cytokinins. These plant growth regulators play a crucial role in initiating and regulating the process of somatic embryogenesis.

Somatic embryogenesis is a valuable technique in plant biotechnology and tissue culture, allowing for the production of whole plants from somatic cells and providing opportunities for genetic manipulation and propagation of plants with desirable traits.

Characteristics of somatic embryogenesis

Somatic embryogenesis is a complex process in plant tissue culture, and it exhibits several distinctive characteristics:

• Bipolar Nature: Somatic embryos are bipolar, meaning they have both a radicle (the embryonic root) and a plumule (the embryonic shoot) end. This bipolarity is essential for the development of complete plants.
• Origin from Two Different Cells: Somatic embryos can be initiated from two different types of cells within the plant tissue, demonstrating the capacity for cellular reprogramming and differentiation.
• Dedifferentiation: Dedifferentiation is a crucial aspect of somatic embryogenesis. It involves the reversion of mature, specialized cells into undifferentiated cells, leading to the formation of callus tissue. This dedifferentiation step is essential for initiating embryogenesis.
• Redifferentiation: After the formation of callus, some cells within the callus have the ability to undergo redifferentiation. This process allows them to develop into differentiated cell types or even whole plantlets, including shoots, roots, and leaves.
• Organogenesis: Organogenesis is the process by which organs, such as shoots, roots, and leaves, are induced and developed in plant tissue culture. It is a critical aspect of somatic embryogenesis, as it leads to the formation of complete plant structures. Organogenesis is also referred to as regeneration, as it involves the regeneration of plant parts.

Overall, somatic embryogenesis is a remarkable phenomenon in plant tissue culture, involving the transformation of differentiated cells into embryogenic structures, followed by the development of complete plants. It has significant applications in plant propagation, genetic engineering, and plant breeding.

Protoplast Culture

Protoplast culture is a specialized technique in plant tissue culture that involves the manipulation and culture of plant cells that have had their cell walls removed.
Here are some key points related to protoplast culture:

• Protoplast Definition: A protoplast is a plant cell that has had its cell wall removed, resulting in a cell without a rigid outer structure. Protoplasts are typically isolated from plant tissues.
• Importance in Hybridization: Protoplast culture is important for the creation of hybrid plants. Hybrids are generated through the fusion of two cells from two different plants. This technique allows for the controlled fusion of protoplasts to create hybrid plants.
• Cybrids: In addition to hybrids, protoplast culture can also be used to create cybrids. Cybrids are generated by fusing the cell of a nucleated plant with an enucleated (lacking a nucleus) cell. This can be a valuable technique in plant biotechnology.
• Protoplast Isolation: Protoplast isolation can be achieved through mechanical or enzymatic methods or a combination of both. These methods break down the cell wall to release the protoplasts.
• Stabilization: To stabilize the plasma membrane of isolated protoplasts and maintain their integrity during culture, substances like calcium chloride (CaCl2) are often used. This helps protect the protoplasts and prevent cell damage.

Protoplast culture is a powerful tool in plant biotechnology, allowing for controlled genetic manipulation and the creation of novel plant hybrids and cybrids. It has applications in plant breeding, genetic engineering, and the study of plant physiology.

Stages of Protoplast Culture

• Protoplast culture is a specialized technique in plant tissue culture that involves isolating and culturing plant cells without their cell walls. Here's a breakdown of the stages involved in protoplast culture:

Stage I: Isolation of Protoplast

Tissue Source: Protoplasts can be isolated from various plant tissues, but leaves are commonly used. The following steps describe the isolation process:

• Sterilization of Leaves:
  • Rinse the leaves with clean water to remove dust and soil particles.
  • Dip the leaves in 70% ethyl alcohol and a 2% solution of sodium hypochlorite for a few minutes.
  • Rinse the leaves several times with sterile distilled water to remove sodium hypochlorite.
• Removal of Epidermis:
  • Gently remove the lower epidermis of the leaves.
  • Cut the leaves into small pieces to facilitate the early isolation of protoplasts.
• Enzymatic Treatment:
  • Enzymatic treatment is performed to break down the plant cell wall. There are two methods:
  • Direct (One-Step) Method:
    • Use a solution containing 0.5% macerozyme or pectinase and 2% cellulase in a 13% mannitol or sorbitol solution at pH 5.4.
    • Incubate at 25-30°C for a few hours.
    • Gently tease the tissue to isolate protoplasts.
  • Sequential (Two-Step) Method:
    • Use 0.5% macerozyme or pectinase along with 0.3% potassium dextran (antioxidant) in a 10% mannitol or sorbitol solution.
    • Place in a water bath at 25°C and shake for 15 minutes.
    • Add fresh enzyme and incubate for an hour.
    • Wash the tissue with 13% mannitol or sorbitol.
    • Add enzyme B (2% cellulase in 13% sorbitol or mannitol) and incubate for a few hours.
• Purification:
  • Filter the solution obtained from enzymatic treatment through a 45 μ Nylon mesh and collect the filtrate.
  • Centrifuge the filtrate at 100g for 1 minute.
  • Discard the supernatant.
  • Wash the pellet three times with 13% mannitol or sorbitol, centrifuging at 100g for 1 minute.
  • Resuspend the pellet in a 20% sucrose solution.
  • Centrifuge at 200g for 1 minute to obtain pure protoplasts in the supernatant, which can be separated using a pipette.

Stage II: Culture

• Protoplast Density: Prepare a protoplast solution with a concentration of 10^5 cells (protoplasts) per milliliter using mannitol or sorbitol.
• Culture Techniques: Protoplasts can be cultured using various techniques, including Bergmann’s culture technique, microchamber technique, or the microdrop method.
• Callus Formation: Initially, callus is formed from the cultured protoplasts.
• Subculture: The callus obtained can be subcultured to promote further growth and development, ultimately leading to the regeneration of plantlets.

Protoplast culture is a valuable technique in plant biotechnology, allowing for genetic manipulation and the generation of new plants with desirable traits. It has applications in plant breeding, genetic engineering, and research on plant physiology.

Applications of Protoplast Culture

• Biochemical and metabolic studies
• Production of hybrid plants (nucleus of two different plants fused)
• Production of cybrid (fusion of nucleated and enucleated cells).
• Genetic manipulation
• To test during sensitivity

Somatic Hybridization

Somatic hybridization is a technique used to produce hybrid plants by fusing protoplasts from two different plant species or varieties.
Here are the stages involved in somatic hybridization:

• Isolation of Protoplasts: Protoplasts are isolated from two different plant species or varieties. This involves breaking down the cell walls to obtain individual protoplasts.
• Mixing of Protoplasts: The protoplasts from the two different plants are mixed together. To facilitate fusion, various fusogen treatments can be used, including treatments with substances like NaNO3, PEG (polyethylene glycol) TR, Ca++, or electrofusion techniques.
• Wall Regeneration: The fused protoplasts, known as heterokaryotic cells, contain nuclei from both parent species or varieties. These cells undergo wall regeneration, forming a cell wall around the fused protoplasts.
• Fusion of Nuclei: In this stage, the nuclei within the heterokaryotic cells fuse together to produce hybrid cells with a combination of genetic material from both parent species or varieties.
• 5. Plating and Colony Formation:
• The hybrid cells are plated on a suitable culture medium, where they divide and form colonies. Each colony represents a group of hybrid cells.
• Selection of Hybrid Colonies: Among the colonies formed, those that exhibit the desired characteristics or traits are selected for further cultivation.
• Subculture and Plantlet Regeneration: The selected hybrid colonies are subcultured to promote their growth and development. Eventually, these colonies can be induced to regenerate into whole plantlets.

Somatic hybridization allows for the creation of plants with a combination of traits from different parent species or varieties. It is a valuable technique in plant breeding and genetic improvement, as it can lead to the development of plants with desirable characteristics, such as disease resistance, improved yield, or unique traits.

Anther and Pollen Culture

Anther and pollen culture are techniques used in plant tissue culture for specific purposes.
Here are some key points about these techniques:

• Objectives:
  • The primary objective of anther and pollen culture is to produce haploid plants, which have a "n" number of chromosomes, resulting in stable genetic variability.
  • Haploid plants are sporophytes with a gametophytic chromosome number and have a single complete set of chromosomes, which can be useful for crop improvement.
• Anther Culture:
  • Anther is the male reproductive organ with a diploid chromosome number.
  • Tetrads of microspores are formed during microsporogenesis, which later become pollen grains.
  • In anther culture, flower buds are sterilized, and anthers are removed carefully to avoid injury. Injury can lead to the development of callus, which produces a mixture of haploids and diploids.
  • The collected anthers are cultured on a suitable medium, and depending on the conditions, they may develop directly into embryoids within 15 days or follow various indirect pathways to produce haploid plantlets.
• Pollen Culture:
  • Pollen culture is preferred over anther culture, although success rates may be lower.
  • About 50 anthers are placed in a medium, and the microspores are squeezed out.
  • The microspores are then filtered through a sieve, and the filtrate containing microspores is centrifuged.
  • The microspores are inoculated on a solid or liquid medium and cultured at specific environmental conditions.
  • The microspores may develop directly into embryoids or follow indirect pathways to produce haploid plantlets.
• Medium and Conditions:
  • Commonly used media for anther and pollen culture include MS (Murashige and Skoog) medium and White's medium.
  • The sucrose concentration in the medium typically ranges from 2% to 3%, but specific concentrations may vary for different plant species, such as dhaturo, tobacco, cereals, or wheat.
• Colchicine Treatment:
  • In some cases, colchicine treatment is used to induce chromosome doubling in haploid plants, resulting in the formation of double haploid plants.

Anther and pollen culture are valuable techniques in plant breeding and genetic research, particularly for generating haploid plants with unique traits or for crop improvement purposes. These techniques provide a means to introduce genetic diversity into breeding programs and accelerate the development of new plant varieties.

Embryo Culture

Embryo culture is a technique used in plant tissue culture, primarily for the production of haploid plants and the recovery of plants from specific crosses. It is particularly valuable in cases where embryos fail to develop due to degeneration of embryonic tissues.
Here are the key steps involved in the procedure of embryo culture:

• Fruit Collection: Start by plucking or collecting the developed fruit from the plant.
• Initial Washing: Wash the collected fruit thoroughly with clean water to remove any contaminants.
• Surface Sterilization: Surface sterilize the fruit using a 0.01% tween 20 solution for about 15 minutes. This step helps eliminate external microorganisms.
• Rinsing: After surface sterilization, rinse the fruit several times with sterile distilled water to remove any residual tween 20.
• Secondary Sterilization: Submerge the fruit in a 0.01% mercuric chloride (HgCl2) solution for 10-15 minutes. This secondary sterilization step is crucial for ensuring the complete removal of any remaining contaminants.
• Final Rinsing: Thoroughly wash the fruit again with sterile distilled water to eliminate any traces of mercuric chloride.
• Isolation of Embryo: Aseptically break open the seed and isolate the embryo. The embryo is the part of the seed that will be cultured.
• Inoculation: Inoculate the isolated embryo onto a culture medium that promotes callus proliferation. This is typically done by placing the embryo on the surface of the medium.
• Callus Formation: After about four weeks of inoculation, callus tissue should begin to form from the cultured embryo.
• Subculture: After eight weeks of inoculation, you can subculture the developing callus onto a medium that supports shoot regeneration.
• Shoot Formation: After approximately 12 weeks from the initial inoculation, you should observe the formation of shoots from the culture.
• Green Callus and Embryoid Formation: Green callus and embryoids (small, immature embryos) will start to appear as the culture continues.

Embryo culture is a powerful technique that allows for the recovery and development of plants from specific crosses and is particularly useful in breeding programs. It can also be employed for the production of haploid plants, which have reduced chromosome numbers and can serve as valuable breeding material. The technique is adapted to the specific needs of different plant species and genotypes, and various modifications can be made to suit different requirements.

Ovary Culture

Ovary culture, also known as gymnogenesis, is a technique in plant tissue culture that serves several objectives:

• Overcoming Pre-Fertilization Barrier: It can be used to overcome barriers to fertilization before the formation of seeds, particularly in cases where natural pollination is inefficient or unsuccessful.
• Overcoming Post-Fertilization Barrier (Embryo Rescue): Ovary culture can also be employed to overcome barriers to embryo development or rescue embryos from ovules or ovaries that would not develop naturally.

Here are some key points about ovary culture:

• Definition: Ovary culture involves the culture of unfertilized or fertilized ovaries to obtain plants.
• Success Rate: The success rate of ovary culture can vary widely, typically ranging from 0.2% to 6%. In some cases, one or two, and rarely up to eight plantlets may originate from each cultured ovary.
• Applications: Ovary culture is often used for in vitro pollination and fertilization or for overcoming post-fertilization barriers, particularly in interspecific or intergeneric crosses. It has been successfully employed in several plant genera, including Brassica, to obtain interspecific hybrids.
• Developmental Stage: In many cases, ovaries are excised at specific developmental stages, such as the zygote stage or the two-celled pro-embryo stage, to ensure normal development in vitro.
• Promotion of Development: Chemical additives, such as indole-3-acetic acid (IAA), fruit juice, or coconut milk, can be incorporated into the culture medium to promote the development of embryos or plantlets.
• Limitations: Ovary culture is not equally successful in all plant species. It has been successfully applied in less than two dozen species, and the success rate can be relatively low.

Ovary culture is a valuable tool in plant breeding and tissue culture, allowing researchers and breeders to overcome specific reproductive barriers and obtain desired hybrids or plantlets that may not develop naturally. While it has its limitations, it has proven effective in certain cases, particularly in obtaining interspecific hybrids for crop improvement and breeding programs.

Applications of Haploids in plant breeding

• Homozygous lines for the production of homozygous plant.
• Hybrid sorting in haploid breeding.
  – Selection of recombinant superior gametes.
• Mutation research
• Gametoclonal variation: variation in gametic chromosomal number
• Cytogenic research: Basic chromosome number
• Evolutionary studies
• Genetic studies

Applications of Plant Tissue Culture

• Production of secondary metabolites. E.g. alkaloids, steroids, phenolics, variety of flavors and perfumes.
• Rapid clonal multiplication or clonal propagation (which also known as micropropagation).
• Production of virus free plant (By thermotherapy, chryotherapy, chemotherapy, and meristem culture).
• Rapid development of homozygous lines by producing haploids (anther culture, ovary culture, etc.)
• Production or recovery of hybrid plants which are difficult to grow in normal conditions. (Embryo rescue, in-vitro pollination).
• Germplasm conservation. (By cryopreservation, and slow growth cultures).
• Genetic modification of plants (somaclonal variation, somatic hybridization and cybridization, gene transfer).
• Creation of genetic maps or genome maps and use of molecular markers.
• Multiplication of the plants of desired size.
• Independent of season so carried out through-out the year.
• Production of synthetic or artificial seeds which are easier to handle, transport and store.