In this tutorial, we have explained ‘What is Plant Tissue Culture & Application in Modern Science’. 

WHAT IS PLANT TISSUE CULTURE?

— The technique of maintaining and growing plant cells, tissues or organs in a sterilized nutrient/culture medium under controlled environmental conditions is known as plant tissue culture. It is one of the latest and most promising methods of crop improvement in those plants where all other conventional methods of breeding fail.

— The plant part or plant material or a single cell or a group of cells that is used for tissue culture is called explant. The explant may be excised root tips, shoot bud, leaf, petiole, inflorescence, anther, ovule, ovary, or embryo. The capacity to generate a whole plant from any plant or explant is called totipotency or cellular totipotency.

— Gottlieb Haberlandt in 1902 started the technique of plant tissue culture when he attempted to culture isolated single cells from leaf mesophyll. This concept of totipotency is further implemented by Steward (1932) when he developed a complete carrot plant from a single cell obtained from the root of a wild carrot. The entirely vegetatively produced descendants of somatic cells are collectively called clone. An individual member of a clone is called ramet.

Plant tissue culture is a technique for studying plant growth, development, and reproduction. Plant tissue culture can also be used to grow new plants that scientists can investigate.

The explants are grown on a solid, semi-solid, or liquid medium which is generally composed of inorganic salts of major and minor elements, vitamins, and sucrose. A medium with these ingredients will be referred to as a basal medium. The solid culture medium contains:

01. Inorganic salts to provide essential macro and micronutrients.

02. Organic nutrients like glucose, sucrose, fructose, etc., to provide energy, amino acid, glycine, and vitamins.

03. Growth hormones (auxins, gibberallins and cytokinins) and agar-agar or gelatin to make the medium solid.

The amount and nature of nutrients in culture medium vary. Standard culture media are now available for most purposes. Most commonly used culture medium includes coconut milk, yeast extract, casein hydrolysate, or bean-seed extract along with plant hormones as growth regulators. Auxin 2,4D (2,4-dichlorophenoxyacetic acid) and Cytokinin BAP (benzylaminopurine) are commonly used growth regulators.

04. An optimum pH (usually 5.7) is also very important. The most extensively used nutrient medium is MS medium which was developed by Murashige and Skoog in 1962. Usually, a gelling agent agar (a polysaccharide obtained from a red algae Gelidium amansi) is added to the liquid medium for its solidification.

Plant tissue cultures are often classified according to the type of in vitro growth viz callus and suspension cultures.

01. Callus Culture

In callus culture, the explant is grown in a nutrient medium (agar-gelled medium). Its cells divide repeatedly and form an unorganized mass of cells, called callus. It takes about 2-3 weaks for the callus to be formed. The callus remains on the surface of the gel nutrient medium. The medium is fortified with both auxin and cytokinin. The common auxin used in callus culture is 2,4-D and the common cytokinin is BAP.

02. Suspension Culture

In suspension culture, single cells or small groups of cells are suspended in a liquid medium. Usually, the medium contains auxin 2, 4-D. The suspension culture is constantly agitated at 100-250 rpm. Agitation of nutrient medium provides constant mixing of medium, breakage of cell aggregates into small cell groups and aeration of the medium. As a result, suspension cultures grow faster than callus cultures.

The procedure of tissue culture involves the following steps:

01. Selection of Explant

The plant tissue to be cultured is excised from the plant from its original location.

02. Sterilization

The explants, the culture vessels, culture media, and the instruments to be used during plant tissue culture are made free from microbes. This is called sterilization. For sterilization, explants are treated with specific antimicrobial chemicals, such as chlorine water or sodium or calcium hypochlorite solution. This is called surface sterilization. The vessels, culture media, and instruments are sterilized with steam, dry heat, or autoclaving at 120° C for 15 to 20 minutes.

03. Transfer of Explants to Culture Medium

The explants are transferred to a sterile culture medium under aseptic conditions either to a solid or fluid culture medium. The setup is kept under aseptic conditions with optimum conditions for growth.

04. Callus Formation

In the culture medium, cells of explant start dividing and form an unorganized mass of cells, called callus. It is formed of parenchymatous cells. These cells are totipotent.

05. Organogenesis and Formation of Plantlets

Under the influence of various concentrations of different hormones in the culture medium, the undifferentiated totipotent parenchymatous cells of the callus differentiate to form shoots and roots, giving rise to miniature plantlets. The fate of growing tissue depends upon the source of selected tissue, i.e., the explants, conditions for growth, and composition of the nutrient medium.

Callus, as well as suspension cultures, can be used for:

01. Obtaining cell biomass which is used for biochemical isolation.

02. Regeneration of plantlets from the callus.

03. Production of transgenic plants which cannot be produced by sexual reproduction.

04. Isolation of protoplasts.

Regeneration is the development of an organized structure like root, shoot or somatic embryo from the cultured cells. The ability of isolated parenchymatous cells of plants in culture to regenerate into complete new plants is called totipotency. Morphogenesis is an interplay of auxins and cytokinins where both must be present in some minimum concentration.

01. Shoot and Root Culture 

Shoot regeneration from the callus cells is promoted by cytokinins like BAP (benzylaminopurine), while root regeneration is accelerated by auxins, like NAA (naphthalene acetic acid). As a matter of fact, shoot and root regenerations occur simultaneously producing plantlets and are controlled by auxin-cytokinin balance.

Callus culture is first kept in the medium containing BAP. Shoot regeneration begins from callus cells. When shoots become 2-3 cm long, they are excised and transferred to auxin containing medium. Roots start regenerating from the lower ends of these shoots to produce plantlets.

02. Somatic Embryo Culture 

Somatic embryo develops from the single diploid somatic cell in a similar fashion as an embryo develops from the zygote. Regeneration of embryo from the somatic cell is induced by a high concentration of auxin 2, 4-D. The mature somatic embryos also germinate into complete plantlets.

03. Establishment of Plantlets in the Field 

The plantlets are removed from culture and are kept under reduced light and high humidity in small pots for a suitable period of time for hardening. The process of hardening makes the plantlets tolerant to the harsh environment in the fields.

04. Meristem Culture 

Cultivation of axillary or apical shoot meristems is called meristem culture. Apical (axillary) meristems are generally free from viruses and other infections. Meristem culture involves the development of the shoot system and regeneration of adventitious roots from the developed shoots. Explants commonly used in meristem culture are shoot tips and nodal segments. The explants are cultured in a medium containing BAP. The plantlets thus obtained are subjected to hardening and then established in the field. Meristem culture is carried out in Sugarcane, Strawberry, Sweet potato, Potato, Banana, Cardamom, Orchids, etc.

The usual method of propagation of commercially important orchids by offshoots is a slow and time consuming process. Now orchids are raised by tissue culture. In several countries, plants are commercially raised by tissue culture.

Tissue culture and genetic engineering are aimed at the selection and cultivation of new plants, resistant to diseases, predators and draught, and which can be grown without fertilizers and pesticides.

There are the following applications of tissue culture in crop improvement:

01. Micropropagation

02 Production of disease-free plants

03. Anther culture and formation of androgenic haploids

04. Embryo culture of successful hybridization

05. Induction and selection of mutants

06. Rapid clonal propagation

07. Somaclonal variations

08. Protoplast culture and somatic cell hybridization

01. Micropropagation 

Micropropagation is the rapid vegetative multiplication of plant material for agriculture, horticulture and forestry. The process is very fast and highly productive. Micropropagation is of great advantage because:

(i). Tissue culture provides rapid multiplication. Under favourable conditions, a small plant bearing five or six leaves is produced within a few weeks which under normal conditions may need several months.

(ii). By tissue culture technique, a large number of offspring or plantlets can be obtained every year. For example, a progeny of 50,000 raspberry plants can be obtained from its meristem culture, whereas only 50 plants a year are formed with conventional cutting techniques.

(iii). The progeny of those plants can be obtained in millions, which multiply with difficulty by conventional methods.

(iv). Cloning can be done throughout the year in a very small space under controlled conditions.

(v). Offspring can be obtained from sterile plants or of rare hybrids of extraordinary characters, e.g., Oil Palm.

Micropropagation is widely used in forestry and in floriculture. It is used in commercial production of ornamental plants like lily, orchids, Eucalyptus, Cinchona and fruit tree like tomato, apple, banana, grapes, Citrus etc.

Micropropagation is achieved by following methods:

a. Multiple Shootlet Production: Shoot tips are used for tissue culture and raising mini-plants. Shoot tips in a culture medium produce multiple buds and each bud grows into a shoot. By using the rooting hormone, the shoot is induced to produce roots.

Micropropagation of Potato, Cardamom, Raspberry, Peach, Almond, Orchids, Bananas, Gerberas Chrysanthemums, Begonias, etc., is being successfully done on a commercial level.

b. Somatic Embryogenesis: The embryos developed from a single somatic cell by tissue culture are known as somatic embryos or embryoids.

In Carrot, Celery and Alfalfa, the somatic embryos can be produced in thousands in a small volume of nutrient medium.

02. Production of Disease-free Plants 

In plants that are propagated vegetatively by roots, bulb, tuber or rhizome, etc., the pathogens (viruses, etc.) are transmitted to the offspring through these propagules. But if plants are raised by tissue culture from the shoot tips of such infected plants, they are free of pathogens.

Healthy plants of Potato, Cassava, Sugarcane, Strawberry, Carnation, Dahlia and many more ornamental plants are grown by tissue culture method.

03. Anther Culture and Formation of Androgenic Haploids 

By anther culture, androgenic haploid embryos or plantlets are obtained from the microspores or pollen. Guha and Maheswari (1964) produced androgenic haploid plants by anther culture of Datura innoxia in culture medium containing kinetin, coconut milk, or grape juice. Now pollen embryos of Wheat, Barley, Rice, Tomato, vegetables, fodder species and ornamental plants (Asparagus) and many other plants (about 250 plants) have been cultured.

The development of pollen into a haploid sporophyte (embryo) in a culture medium is known as male parthenogenesis or androgenesis. If pollen grains are treated with colchicine before culture, homozygous diploid embryos are formed (colchicine stops the cell division after chromosome separation and leads to chromosome doubling).

— Anthers from F₁ plants (obtained by crossing two or more lines) are cultured to obtain haploid plants.

— The chromosome number of these haploid plants is doubled by using colchicine to obtain homozygous plants. The progeny from these plants are then subjected to selection to isolate superior homozygous lines.

— This approach has been successfully used to develop several varieties.

— Haploids are very important in plant breeding because: 

➢ They have a single set of chromosomes, so even a very small change or mutation can be detected in haploids.

➢ These are used to produce homozygous diploids (by colchicine treatment) and these homozygous diploids are used as parents in crossing programs.

► Significance of Haploid Plants: Such plants have the following significance:

(i). In haploid plants, mutations (spontaneous or induced) can express themselves and can be easily detected because haploids have only a single set of genes. In diploids, a mutation is not easily detectable because they are heterozygous.

(ii). Homozygous diploids can be obtained in a single generation by treating a haploid cell in a culture medium with colchicine.

(iii). When used on a large scale in hybrids, it combines the advantages of recombination, segregation, and fixation.

► Ovule Culture: This technique is applied to raise hybrids which normally fail to develop due to abortion of the embryos at an early stage. Ovules are excised from the ovary and cultured in the basal medium. The loss of a hybrid embryo due to premature abscission of fruits may be prevented by ovule culture. In some cases of adding fruit or vegetable, juice increases initial growth.

► Ovary Culture: Ovary culture has helped in raising interspecific hybrids between sexually incompatible species, Brassica campestris and B. oleracea. Ovaries are excised from the flowers and cultured at the zygote or two-celled proembryo stage for obtaining normal development in a culture medium. Coconut milk when used as a supplement to the medium promotes the formation of fruits that are larger than those formed in vivo. In Anethum, multiple shoots are obtained by inducing polyembryony by adding kinetin in the medium.

04. Embryo Culture 

Embryo culture is the excision of young embryos from the developing seeds and their in vitro cultivation on a nutrient medium. It is utilized in some crop improvement programs to obtain interspecific hybrids, overcoming dormancy and propagation orchids.

Interspecific or intergeneric hybrids are usually sterile because of embryo mortality and seed collapse. In such cases, the young hybrid embryo is excised from the ovary and cultured in vitro. This is described as embryo rescue.

► Application of Embryo Culture: Embryo culture allows for to complete development of such embryos which are unable to develop in vivo because of the following reasons:

(i). Degeneration of Endosperm: In some interspecific crosses, the endosperm of the developing hybrid seeds degenerates early. The young embryos, unable to derive nourishment from the degenerated endosperm also collapse, causing sterile hybrids. In such cases, the embryo culture technique provides hybrid seedlings.

(ii). Dormant seeds: Inhibitors present in endosperm and other parts of seeds do not allow the embryos to grow. Embryos of such seeds can be excised and grown over a culture medium to form seedlings. It eliminates the action of inhibitors and dormancy.

(iii) Seed sterility: It is caused by noncompletion of embryo development due to early fruit ripening (e.g., Prunus, early ripening varieties) or death of embryo caused by mutations of embryo covering structures (e.g., macapuno, coconut). A young embryo of such seeds is taken out and cultured over a nutrient medium where it forms the seedling.

(iv). Lack of Stored Food: Seeds of some plants (like orchids) lack stored food. Embryo culture in such cases produces seedlings.

(v). Recalcitrant Dormancy: In orchids, bananas, and other plants with a long period of dormancy, embryo culture is used for rapid clonal propagation.

(vi) Rare plants: The technique is useful in the multiplication of some rare plants, e.g., makapuno nut. In intergeneric and interspecific hybrids, embryo abort at earlier or later stages. By using embryo culture we can save rare hybrids. In this technique, an embryo is excised at a younger stage and is cultured in a culture medium. So, maintenance of rare hybrids is by embryo culture.

► Examples of Embryo Culture: Embryo culture has been widely used in rescuing embryos of hybrid crosses and producing hybrids. For example:

(i). Hybrids of common bean (Phaseolus vulgaris) and wild bean (P. angustissimus) were developed by this method. The hybrid progeny is disease-resistant and has many other desirable traits.

(ii). Hybrids of cultivated tomato, Lycopersicum esculentum and wild variety, Lycopersicum peruvianum have been developed by this method. These interspecific hybrids are disease resistant and have improved vitamin C content.

(iii). In 1986, Khush and Jena have successfully transferred genes for brown plant-hopper resistance from Oryza officinalis to O. sativa, through hybridization and embryo rescue.

(iv). In India, success has been achieved through embryo rescue in the hybridization of two jute species, Corchorus olitorius and C. capsularis. The hybrids have improved quality and showed resistance to pests and diseases.

05. Induction and Selection of Mutants 

Cells can be grown in suspension cultures by using a liquid culture medium. The cultures can be exposed to continuous shaking by a mechanical shaker. As a result, the culture consists of isolated colonies. The isolated individual cells can be transferred to separate culture plates where clones of cells are grown by adding chemical mutagens into the medium for various traits. When herbicides or toxins are added to the culture medium, the colonies or clones of cells that are resistant survive and others get perished. The plants can be raised from these tolerant cells for agriculture purposes. Thus, by tissue culture technique, mutations can be introduced in the cultures and resistant mutants can be selected to produce resistant varieties.

06. Rapid Clonal Propagation 

Clone means exact carbon copy. It can be defined as a group of cells or individuals derived from a single cell or parent through asexual reproduction. Being derived from a single cell, all the cells of a clone have identical genotypes and exhibit little or no differences. Cloning is the process of producing many identical cells or clones. Cloning is based on the totipotency of plant cells. This way, all the cells in a callus or in suspension culture are clones because these are derived from a single explant by mitosis. Therefore, all plantlets regenerated from the cells of a single callus are clones. These plantlets provide a means of rapid propagation.

Many orchid plants that bear beautiful flowers are cloned plants. Scientists have genetically engineered agronomically important crop plants. Their rapid production is achieved by clonal propagation. Microorganisms produce clones in nature and are being used in biotechnology.

07. Somaclonal Variations

Somaclonal variations are genetic variations observed in those plants that are raised from the callus by tissue culture. If these variations are of economic value, because they may have, induced tolerance for herbicides, pests, diseases, or environmental stresses or are helpful to overcome male sterility, such plants are selected and multiplied. Somaclonal variants are known in: 

(i). Wheat: These are resistant to rust and have a tolerance to high temperatures.

(ii). Rice: These are resistant to Tungro virus and leaf hoppers.

(iii) Potato: These have high protein content.

(iv). Tomato: These have increased shelf life.

08. Protoplast Culture and Somatic Cell Hybridization

Protoplasts are the naked plant cells whose cell wall is dissolved by the enzymes cellulase and pectinase. Protoplasts can be isolated from leaves, callus, suspension cultures and from pollen grains. These can be grown on agar containing nutrients.

► Genetic Manipulation and Somatic Cell Fusion 

Fusion of plant cells is not possible because of a thick cellulosic wall. The plant cells are also held together by the middle lamella. Therefore, the preparation of the protoplast is an essential step in tissue culture.

The protoplast culture is used for the following purposes:

● Protoplasmic Fusion or Somatic Cell Hybridization: A hybrid produced by the fusion of somatic cells of two varieties or species is called a somatic hybrid

. The process of producing somatic hybrids is called somatic hybridization. In plants, this is achieved by protoplast fusion. The protoplasts from different varieties of the same or different species are made to fuse by chemofusion (through sodium nitrate or PEG = Polyethylene glycol) or by electrofusion (high frequency alternating electric field with short current phases). It results in hybrid protoplasts. The somatic hybrid may have a synkaryon (single fused nucleus) or heterokaryon (having two unfused nuclei).

This provides chances for hybrid formation between species of Brassica, Nicotiana, Petunia and Solanum and between Rice and Carrot. In 1978, Tomato and Potato protoplasts were fused. The resulting hybrid is called Pomato. Likewise, Brourato is an intergenic hybrid between Brinjal and Tomato. It is of great scientific interest and agronomic importance of raising hybrids between distantly related organisms. 

► Applications of Protoplasmic Fusion or Somatic Cell Hybridization

This technique can be used for:

1. Production of hybrids between species that cannot be produced by sexual hybridization.

2. Gene transfer and formation of transgenic plants.

3. Transfer of cytoplasm.

4. Production of allotetraploids.

● Production of Transgenic Plants:  A gene that is inserted in the cell by genetic engineering is called a transgene. An organism that contains and expresses the transgene is known as a transgenic organism. Transgene or transgenes are introduced into the protoplasts. The cells containing transgenes are selected in vitro and isolated. The transgenic cells are then grown in a culture medium where the plantlets regenerate from these cells. These transgenic plantlets are grown into transgenic plants.

● Obtaining Clones: Protoplasts are grown to obtain their clones which can be used for various genetic manipulations.

● Clone Selection: Clones derived from leaf cell protoplasts are selected to give rise to plants with particular useful character.

The conventional agricultural produce of cereals, pulses, vegetables, and fruits would not be able to meet the demand for food for the increasing human and animal populations. Already more than 25% of the human population is suffering from hunger and malnutrition. The shift from a grain to meat diet is also creating more demand for cereals because it takes 3-10 kg of grains to produce 1 kg of meat from animal farming. Even a 250 kg cow produces just 200 g of protein per day while it eats 20–30 kg of food. Therefore, one of the alternative sources of proteins for animal and human nutrition is single cell protein (SCP).

Microbes have been used for a long at the domestic as well as the commercial level in the preparation of fermented foods like cottage cheese, curd, butter, idli, etc. Spirulina and mushrooms have long been used as human food.

► Substrate for Single Cell Protein Production 

● Microbic biomass: It is being produced on a low-cost substrate that can be used for feed or for human consumption. They can be grown easily on materials like wastewater from potato processing plants containing starch, straw, molasses, animal manure, and even sewage to produce large quantities of food rich in proteins, minerals, fats, carbohydrates and vitamins. As a matter of fact, such utilization also reduces environmental pollution.

● Cow versus Microbes:  It has been calculated that a 250 kg cow produces only 200 g of protein per day. In the same period, 250 g of a microbe like Methylophilus methylotrophus, because of its high rate of biomass production and growth, can produce 25 tonnes of protein from waste materials. The common sources of single cell proteins are yeasts and other fungi like Fusarium graminearum.

► Some Common Microbes as SCP Producers

1. Cyanobacteria: Spirulina

2. Bacteria: Methylophilus methylotrophus

3. Yeasts: Candida utilis

4. Filamentous Fungi: Fusarium graminearum

► Significance of Single Cell Protein  

01. SCP can be used as a protein-rich supplement to the human diet.

02. This will help in bridging the gap between the demand and supply of protein for the human diet and reduces the pressure on agriculture.

03. Use of organic wastes and industrial effluents in raising SCP will reduce environmental pollution.