Tissue Culture – A Sustainable Approach to Explore Plant Stresses

Microbiology is one of the Biology Topics that involves the study of microorganisms, including bacteria, viruses, and fungi.

Plant Tissue Culture – Techniques, Process, and its Advantages

The technique of maintaining and growing cells, tissues, or organs, especially on artificial medium in suitable containers under controlled environmental conditions is known as tissue culture. In this technique, culturing of plant seeds, organs, explants, tissue cells, or protoplasts on well chemically defined systematic nutrient media.

The tissue culture technique was first practiced by Haberlandt (1902). Hanning and Harrison (1907) developed the technique. The first successful attempt was made by White (1932) in the case of Tomato root. Skoog and Miller (1957) observed that the growth and morphogenesis were controlled by hormones, auxin (root formation), and cytokinin (shoot formation) in tissue culture. Steward (1963), Helperin, and Wetherell (1964) successfully developed somatic embryos from young anther cells of Datura. In India, work in tissue culture started at the Department of Botany of Delhi University in 1960.

The details of the plant tissue culture technique and its utilization are emphasized here as follows.

Types of Tissue Culture

Plant tissue cultures are often classified according to the type of in vitro growth, these are Organ Culture, Callus Culture, Embryo Culture, Suspension Culture, Protoplast Culture, Endosperm Culture, Anther Culture, Ovule Culture etc.

1. Callus Culture:
In callus culture, the explant undergoes cell division and forms an unorganized and undifferentiated mass of cells called Callus. The callus culture is maintained on an agar medium. The medium generally contains auxin like 2, 4-D, and often a Cytokinin like BAP (Benzyl-aminopurine).

2. Organ Culture:
A particular organ (Reproductive or somatic) like root or shoot meristems or leaf primordia or floral buds is isolated and cultured. The shoot and root culture are generally controlled by auxin-cytokinin balance, usually an excess of auxin promotes root culture, whereas that of cytokinin promotes shoot culture.

3. Embryo Culture:
An embryo develops within the female gametophyte by sexual reproduction which then undergoes several developmental stages. It can be isolated aseptically from the bulk of maternal tissues of ovule, seed, or capsule and cultured in vitro under aseptic and controlled physical conditions to grow directly into plantlets. In this case, young embryos are taken out from developing seeds and grown on a culture medium to form seedlings and then young plants.

During interspecific hybridization, often the embryo dies quite early so that no mature seeds can be obtained. This problem can be overcome by embryo culture. Seeds of orchids lack stored food. Embryo culture in such cases allows seedling development from most of the embryos. Some rare plants reproduce through seeds with great difficulty. Embryo culture is useful in the multiplication of such plants e.g., makapuno nut.

The culture of the embryo may be divided into the following categories:
(i) Culture of Mature and Intact Seed Embryo:
The aim of this study is to analyze the various parameters of embryonic growth and the metabolic and biochemical aspects of dormancy and germination.

(ii) Culture of Surgically Dissected Embryo:
The mature seed embryo can be dissected surgically into a number of segments. Such embryo segments are cultured to analyze the relationship of different parts of the embryo to its final form in culture.

(iii) Culture of Immature Embryos or Pro-embryos:
The term pro embryo means the early developmental stages of the embryo that precede cotyledon initiation. Globular and heart-shaped stages of embryos are appropriately called pro embryos. The objective of such a culture is to understand the control of differentiation and the nutritional requirements of such progressively developing embryos.

(iv) Culture of Intact Seed Containing Undifferentiated Embryo:
Each fruit of an orchid plant develops several thousand tiny seeds which contain morphologically undifferentiated embryos. These embryos are the spherical mass of tissue lacking both radicle and plumule.

(v) Culture of Adventive Embryos form Polyembryonic Seeds:
Besides the zygotic embryo is produced like lemons and oranges. Such additional abortive embryos can be exploited in culture for clonal propagation.

(vi) Culture of Inviable or Abortive Embryos:
In many inter-specific or inter-generic breeding experiments, sometimes inviable or abortive embryos may develop due to unsuccessful crosses. As a result, the non-viable seeds do not germinate normally. But it is now possible to raise a hybrid plant by culturing the inviable embryos in vitro.

Principles of Embryo Culture

The underlying principle of the method is the aseptic excision of the embryo and its transfer to a suitable nutrient medium for development under optimum culture conditions. In general, it is relatively easy to obtain pathogen-free embryos, since the embryo is lodged in the sterile environment of the ovule or seed or capsule, or fruit. So surface sterilization of the embryos as such is not necessary. Thus the entire seeds or fruits containing the ovule are surface sterilized and the embryos are then aseptically separated from the surrounding tissues. The most important aspect of embryo culture is the crucial selection of the medium, necessary to sustain the growth of the embryos. The changing nutritional requirement for successful embryo culture has often meant transferring the embryo from one medium to another for optimum growth. Monnier devised a culture method that allowed the uninterrupted growth of globular embryos to maturity. By this method, embryos can be grown in both solid and liquid mediums at the same time.

The composition of both media is different. Monnier also emphasized on the importance of obtaining uninjured embryos with their suspensor for successful culture. Suspensor is important for the growth of immature embryos. But embryos selected at later stages do not require the attached suspensor. In culture, the embryos are not induced to form callus tissue but they are allowed to form a plantlet. After the embryos have grown into plantlets in vitro, they are generally transferred to sterile soil and grown to maturity in the greenhouse.

The Procedure of Embryo Culture
The following protocol for embryo culture is based on the method used for Capsella bursapastoris. With modification, this basic protocol should be applicable to embryo culture in general. The steps are given below:

• Capsules in the desired stages of development are surface sterilized for 5-10 minutes in 0.1% HgCl₂, either in a closed small room previously illuminated by UV lamps or in a Laminar airflow.
• Wash repeatedly in sterile water.
• Further operations are carried out under a specially designed dissecting microscope at a magnification of about 90X. The capsules are kept in a depression slide containing a few drops of liquid medium.
• The outer wall of the capsule is removed by a cut in the region of the placenta, and the halves are pushed apart with forceps to expose the ovules.
• A small incision in the ovule followed by slight pressure with a blunt needle is enough to free the & embryos.
• The excised embryos are transferred by micro-pipettes or small spoon-headed spatula to standard 10 cm Petri dishes containing 25 ml of solidified standard medium. Usually, 6-8 embryos are cultured in a Petri dish.
• The Petri dishes are sealed with cello tape to prevent desiccation of the culture.
• The cultures are kept in a culture room at 25°C and given 16 hrs, illumination by a cool white fluorescent tube.
• Subcultures into fresh medium are made at approximately four weeks intervals.

Germination Embryo in Culture
A lot of cellular, physiological, and biochemical changes take place during embryo development from the zygote to the fully formed embryo stage. After its full-term development, the embryo becomes dehydrated and enters a phase of metabolic quiescence and developmental arrest (dormancy) which may last from a few days to several months or even years.

During this stage, the embryo is normally incapable of germination. Embryos of mangroves and some viviparous varieties of cultivated plants (e.g., Sechium edule) germinate while still attached to the parent plant. They are able to bypass the stage of dormancy. A similar phenomenon has been observed when excised immature plant embryos are grown in vitro. In culture, excised immature embryos do not proceed further than the embryogenic development for its maturation and start to germinate. The main objective of culturing immature embryos is to stimulate normal embryological development in order to understand the factor(s) that regulate the orderly development of embryos in nature.

4. Suspension Culture

In suspension culture single cells or small aggregates of cells grow and multiply while suspended in an agitated liquid medium. From suspension culture, cell clones may be developed. Production of secondary metabolites and mutagenetic studies can be done in suspension culture.

Principles of Suspension Culture
Callus proliferates as an unorganized mass of cells. So it is very difficult to follow many cellular events during its growth and developmental phases. To overcome such limitations of callus culture, the cultivation of free cells as well as small cell aggregates in a chemically defined liquid medium as a suspension was initiated to study the morphological and biochemical changes during their growth and developmental phases.

To achieve an ideal cell suspension, most commonly a friable callus is transferred to an agitated liquid medium where it breaks up and readily disperses. After eliminating the large callus pieces, only single cells and small cell aggregates are again transferred to a fresh medium and after two or three weeks a suspension of actively growing cells is produced.

Methods of Suspension Culture

• Take 150/250 ml conical flask containing autoclaved 40/60 ml liquid medium.
• Transfer 3-4 pieces of pre-established callus tissue (approx, wt. 1 gm. each) from the culture tube using the spoon-headed spatula to conical flasks.
• Flame the neck of the conical flask, and close the mouth of the flask with a piece of aluminium foil or a cotton plug. Cover the closure with a piece of brown paper.
• Place the flasks within the clamps of a rotary shaker moving at 80-120 rpm (revolution per minute).
• After 7 days, pour the contents of each flask through the sterilized sieve pore, diameter 60µ – 100µ, and collect the filtrate in a big sterilized container. The filtrate contains only free cells and cell aggregates.
• Allow the filtrate to settle for 10-15 min, or centrifuge the filtrate at 500 to 1,000 rpm and finally pour off the supernatant.
• Resuspend the residue cells in a requisite volume of fresh liquid medium and dispense the cell suspension equally in several sterilized flasks (150/250 ml). Place the flasks on the shaker and allow the free cells and cell aggregates to grow.
• At the next subculture, repeat the previous steps but take only one-fifth of the residual cells as the inoculum and dispense equally in flasks and again place them on a shaker.
• After 3-4 subcultures, transfer 10 ml of cell suspension from each flask into a new flask containing 30 ml of fresh liquid medium.
• To prepare a growth curve of cells in suspension, transfer a definite number of cells measured accurately by a hemocytometer to a definite volume of liquid medium and incubate on a shaker.
• Pipette out a very little aliquot of cell suspension at short intervals of time (1 or 2 days interval) and count the cell number. Plot the cell count data of a passage on graph paper and the curve will indicate the growth pattern of the suspension culture.

Importance of Suspension Culture:

• The culture of single cells and small aggregates in a moving liquid medium is an important experimental technique for a lot of studies that are not correctly possible to do from the callus culture. Such a system is capable of contributing much significant information about cell physiology, biochemistry, and metabolic events at the level of individual cells and small cell aggregates.
• It is also important to build up an understanding of organ formation or embryoid formation starting from single-cell or small-cell aggregates.
• Suspension culture derived from medicinally important plants can be studied for the production of secondary metabolites such as alkaloids and a considerable amount of industrial effort is being placed on the exploitation and expansion of this area.
• Mutagenesis studies may be facilitated by the use of cell suspension cultures to produce mutant cell clones from which mutant plants can be raised.
• Plants could be raised from the mutant cell clones and the mutant plants are selected from the population either by morphological differences or by metabolic/biochemical differences. The selected plants can then be grown on and propagated further to produce a mutant population for evaluation studies.

Differences between Callus Culture and Suspension Culture:

Callus Culture	Suspension Culture
1. In this culture, cell division in explants forms a callus. Callus is an irregular, unorganized, and undifferentiated mass of actively dividing cells.	1. This culture consists of single cells and small groups of cells suspended in a liquid medium.
2. The culture is maintained in an agar medium.	2. The culture is maintained in a liquid medium.
3. The medium contains growth regulators like auxin, 2-4D, cytokinin, etc.	3. The medium contains growth regulators like’ auxin, 2-4D.
4. Callus is obtained within 2-3 weeks.	4. Suspension cultures grows much faster than callus culture.
5. It does not need to be agitated.	5. It must be constantly agitated at 10-250 rpm.

5. Protoplast Culture

The plant cells without cell walls are called protoplasts. Enzymes like pectinase and cellulase are used to isolate protoplasts from cells by digesting cell walls. The protoplasts are cultured on a suitable medium where they regenerate cell walls and begin to divide to produce plantlets. Developing protoplasts and the formation of somatic hybrids are possible with this technique. Genetic manipulations can be carried out more rapidly when plant cells are in a protoplast state. New genes can be introduced. There is a distinct possibility of the development of new crop plants, e.g., Pomato (fusion of potato and tomato).

Protoplasts are naked plant cells without a cell wall, but they possess plasma membranes and all other cellular components. They represent the functional plant cells but lack a barrier, the cell wall. Protoplasts of different species can be fused to generate a hybrid and this process is referred to as somatic hybridization (or protoplast fusion). Hybridization is the phenomenon of fusion of a normal protoplast with an enucleated (without nucleus) protoplast that results in the formation of a hybrid or cytoplast (cytoplasmic hybrids).

Isolation of Protoplasts:
Protoplasts are isolated by two techniques – The mechanical method, Enzymatic method.

• Mechanical Method: Protoplast isolation by mechanical method is a crude and tedious procedure. This results in the isolation of a very small number of protoplasts.
• Enzymatic Method: Enzymatic method is a very widely used technique for the isolation of protoplasts. The advantages of the enzymatic method include good yield of viable cells, and minimal or no damage to the protoplasts.

Sources of Protoplasts:
Protoplasts can be isolated from a wide variety of tissues and organs that include leaves, roots, shoot apices, fruits, embryos, and microspores.

Isolation of Protoplasts from Leaves:
Leaves are most commonly used, for protoplast isolation, since it is possible to isolate uniform cells in large numbers.
The procedure broadly involves the following steps:

• Sterilization of leaves.
• Removal of the epidermal cell layer.
• Treatment with enzymes.
• Isolation of Protoplasts.

Besides leaves, callus cultures and cell suspension cultures can also be used for the isolation of protoplasts. For this purpose, young and actively growing cells are preferred.

Culture of Protoplasts
The very first step in protoplast culture is the development of a cell wall around the membrane of the protoplast. This is followed by the cell divisions that give rise to a small colony. With suitable manipulations of nutritional and physiological conditions, the cell colonies may be grown continuously as cultures or regenerated into whole plants. Protoplasts are cultured either in semisolid agar or liquid medium. Sometimes, protoplasts are first allowed to develop cell walls in a liquid medium and then transferred to an agar medium.

Regeneration of Protoplasts
Protoplast regeneration which may also be regarded as protoplast development occurs in two stages Formation of the cell wall, Development of the callus/whole plant.

1. Formation of Cell Wall:
The process of cell wall formation in cultured protoplasts starts within a few hours after isolation which may take two to several days under suitable conditions. As cell wall development occurs, the protoplasts lose their characteristic spherical shape.

2. Development of Callus/Whole Plant:
As the cell wall formation around protoplasts is complete, the cells increase in size, and the first division generally occurs within 2-7 days. Subsequent divisions result in small colonies, and by the end of the third week, visible colonies (macroscopic colonies) are formed. These colonies are then transferred to an osmotic-free (mannitol or sorbitol-free) medium for further development to form callus. With induction and appropriate manipulations, the callus can undergo organogenic and embryogenic differentiation to form the whole plant finally.

Importance of Protoplast Cultures

• The isolation, culture, and fusion of protoplasts is a fascinating field in plant research.
• Protoplast isolation and their cultures provide millions of single cells (comparable to microbial cells) for a variety of studies.
• The protoplast in culture can be regenerated into a whole plant.
• Hybrids can be developed from protoplast fusion.
• It is easy to perform single-cell cloning with protoplasts.
• Genetic transformations can be achieved through the genetic engineering of protoplast DNA.
• Protoplasts are excellent materials for ultra-structural studies.
• Isolation of cell organelles and chromosomes is easy from protoplasts.
• Protoplasts are useful for membrane studies (transport and uptake processes).
• Isolation of mutants from protoplast cultures is easy.

Selected Examples of Plant Species Regenerated from Protoplasts:

Category	Plant Species
1. Cereals	Oryza sativa Zea mays Hordeum vulgare
2. Vegetables	Cucumis sativus Brassica oleracea Capsicum annuum
3. Woody trees	Larix eurolepis Coffea canephora Prunus avium
4. Ornamentals	Rosa sp. Chrysanthemum sp. Pelargonium sp.
5. Tubers and roots	Beta vulgaris Ipomoea batatas
6. Oil crops	Helianthus annuus Brassica napus
7. Legumes	Glycine max

6. Endosperm Culture

Tissue culture methods are also used for culturing endosperm. Triploid plants produced from endosperm culture are used for the production of seedless fruits (e.g., apples, bananas, etc.). This technique involves the following steps:

• The immature seeds are dissected under aseptic conditions. Endosperms along with embryos are excised.
• The excised endosperms are cultured on a suitable medium and embryos are removed after initial growth.
• During endosperm culture, there is a restoration and the loss of the function of the Dek 1 gene and this is a reversible process.
• The initial callus phase is developed.
• The shoots and roots may develop and complete triploid plants are formed for further use.

7. Anther Culture

The anthers are removed and kept in a culture medium having sucrose, vitamins, auxins, and minerals. After 4-6 weeks, the anthers produce a large number of haploid embryoids. This is also known as androgenic haploid culture or pollen grain culture. The embryoids grow to form haploid plants which are sexually sterile. They are changed to homozygous diploids by using colchicine. 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. Homozygous diploids are used as parents in the crossing programme.

8. Ovule Culture

Fertilized ovule can also be cultured in cases where embryos abort very early and the culture of the embryo is not possible due to difficulty in excision at a very early stage. Ovules can easily be excised from the ovary and cultured on the used basal medium. In most cases, ovaries are often cultured either for in vitro pollination, fertilization, or embryo rescue. In the case of interspecific or intergeneric crosses, the ovaries are excised at the zygote stage or at the two-celled proembryo stage and cultured on synthetic media. Fruits can be obtained by culturing ovaries on synthetic media containing coconut milk, auxin, or other specific requirement.

Requirements for Tissue Culture

• A tissue culture laboratory
• Hot-air oven
• Refrigerator
• Autoclave
• Weighing balance
• Working table
• Inoculation chamber
• Glasswares like flasks; culture tubes, measuring cylinders, etc.
• Constituents for nutrient medium include organic and inorganic substances, hormones, vitamins, amino acids, etc.
• Culture Room
• Distillation plant
• Other instruments like scalpels, forceps, scissors, etc.

Culture Medium

A culture medium composed of inorganic salts on iron sources, vitamins, amino acids, hormones, and a carbon source. Many works like Murashige and Skoog, White, Gamborg, Nitsch and Nitsch, etc. have proposed the composition of a nutrient medium for the growth of plant tissue. The composition of a commonly used (Murashige and Skoog – MS medium) medium is mentioned below.

Murashige and Skoog (MS) Medium:

Constituent	Amount (mg/l)
NH4NO3	1650
KNO3	1900
CaCl2, 2H2O	440
MgSO4, 7H2O	370
KH2PO4	170
KI	0.83
H3BO3	6.2
MnSO4, 4H2O	22.3
ZnSO4, 7H2O	8.6
Na2MoO4, 2H2O	0.25
CuSO4, 5H2O	0.025
CoCl2, 6H2O	0.025
Na2EDTA, 2H2O	37.30
FeSO4, 7H2O	27.80
Sucrose	30,000
Inositol	100
Nicotinic Acid	0.50
Pyridoxine, HCl	0.50
Thiamine, HCl	0.10
Glycine	2
Indole Acetic Acid	0.3 – 3
Kinetin	1 – 2

Culture Technique

The procedure of tissue culture involves the following step:

1. Selection of Explant:
An explant is the plant part that is excised from its original location and used for initiating a culture. Thus, the explant to be cultured is excised from the plant selected for the presence of desired traits.

2. Sterilization:
The explants, culture room, culture vessels, culture media and the instruments (e.g., forceps, scalpels, spatula, etc.) to be used are made free from microbes. This is called sterilization. For sterilization, the explant is treated with specific antimicrobial chemicals, such as Calcium or Sodium hypochlorite solution. This is called Surface sterilization. The vessels, culture media, and instruments are sterilized with steam, dry heat, or autoclaving at 121°C for about 20 minutes. The culture room and larger transfer areas can be sterilized through UV-radiation exposure.

3. Transfer of Explant to Culture Medium or Inoculation:
The explants are transferred to a sterile culture medium under aseptic conditions either to a solid or liquid culture medium. The setup is kept under aseptic conditions with optimum conditions for growth.

4. Incubation:
Culture vials are incubated in an incubation room under proper photoperiod at optimum temperature and relative humidity.

5. Callus Formation:
In the culture medium, cells of the explant start dividing and form an unorganized mass of cells, called a Callus. It is formed of parenchymatous cells.

6. Organogenesis and Formation of Plantlets:
Under the influence of various concentrations of different hormones in the culture medium, the undifferentiated parenchymatous cells of the callus differentiate to form shoots and roots, giving rise to miniature plantlets. The fate of growing tissue depends upon the explant, conditions for growth, and composition of the nutrient medium.

7. Hardening:
Cultured plants are grown in carefully controlled environments providing optimal conditions for growth. But these conditions in nature are variable. Thus before ex vitro transfer, tissue-cultured plantlets need to be acclimatized to environmental conditions. Therefore they are transferred to pots and exposed to high humidity and reduced light thus making them capable of surviving uncontrolled and harsher ex vitro environment. This is called hardening or acclimatization. Hardened plantlets are then ready to be transferred to fields or greenhouses.

Callus Culture from Carrot Root

Callus tissue can be induced from different plant tissues of many plant species. Carrot is a highly standardized material. The procedure is

• A fresh tap root of carrot is taken and washed thoroughly under running tap water.
• The tap root is then dipped into 5% ‘Teepol’ for 10 minutes and then the root is washed. The carrot root, sterilized forceps, scalpels, other instruments, autoclaved nutrient medium, and Petri dishes are then transferred to the inoculation chamber. Throughout the process of forceps, scalpels must be kept in 95% ethanol and flamed thoroughly before use.
• The tap root is surface sterilized by immersing it in 70% ethanol for 60 seconds, followed by 20-25 minutes in sodium hypochlorite solution.
• The root is washed thoroughly with sterile distilled water to remove the hypochlorite completely. The carrot is then transferred to a sterilized Petri dish containing filter paper. A series of transverse slices 1 mm in thickness is cut from the root using a scalpel.
• Each piece is transferred to another sterile Petri dish. An area of 4 mm2 across the cambium is cut from each piece and transferred to a nutrient medium in a flask or tube carefully under aseptic conditions.
• The inoculated flasks are transferred to the culture room and incubated in the dark at 25°C.
• Usually, after 4 weeks in culture, the explants incubated on the medium will form a mass of callus. The whole callus mass is taken out aseptically on a sterile Petri dish and divided into two or three pieces.
• Each piece of callus tissue is transferred to a tube containing fresh medium. Prolonged culture of carrot tissue produces large callus.

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. Plant tissue culture 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.
• The production of progenies 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.

Plant tissue culture relies on the fact that many plant cells have the ability to regenerate a whole plant (totipotency). 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.

Applications of Plant Tissue Culture in Plant Science

Plant tissue culture is used widely in plant science, forestry, and horticulture. Applications include:

• The commercial production of plants is 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 characteristics, e.g., herbicide resistance/tolerance.
• Large-scale growth of plant cells is the liquid culture in bioreactors for the 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 cross-pollinate distantly related species and then tissue culture the resulting embryo which would otherwise normally die (Embryo Rescue).
• For the production of doubled monoploid plants from haploid cultures to achieve homozygous lines more rapidly in breeding programs, usually by treatment with colchicine which causes a doubling of the chromosome number.
• 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 virus-infected stock, such as potatoes and many species of soft fruit. Production of identical sterile hybrid species can be obtained.

Importance of Tissue Culture

The importance and application of plant cell and tissue culture in plant science are vast and varied. The principal applications are discussed briefly under the following sub-headings.

1. Micropropagation:
It is the rapid vegetative multiplication of plant material for agriculture, horticulture, and forestry. A large number of plantlets may be obtained within a short period.

2. Production of Disease Free Plants:
In plants that are propagated vegetatively by roots, bulbs, tuber, etc., the pathogens 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 from pathogens. Healthy plants of potato, sugarcane, and Dahlia may be obtained by this method.

3. Embryo Culture:
Embryo culture allows the complete development of such embryos which are unable to develop in vivo for various reasons.

4. Somatic Hybridization:
A hybrid produced by the fusion of somatic cells of two varieties or species is called a somatic hybrid. In plants this is achieved by the protoplast fusion, e.g., Pomato is a somatic hybrid between tomato and potato.

When a hybrid is produced by the fusion of somatic cells of two species, it is known as somatic hybrid, the overall process is called somatic hybridization. Here the production of protoplasts is done by using a combination of pectinase and cellulose.

5. Production of Haploid Plants:
This is done by pollen culture in a suitable medium. Haploid plants are important for the production of homozygous diploids, mutation studies, etc.

6. Rapid Clonal Propagation:
The multiplication of genetically identical copies of a variety by asexual reproduction is called clonal propagation. A plant population derived from a single donor plant in tissue culture constitute a clone. Many orchid plants that bear beautiful flowers are cloned plants.

7. Production of Somadonal Variants:
Parental variations present among plant cells of a culture are called somaclonal variations. Variants are selected through tissue culture. Such variants may show useful characteristics like resistance to disease, resistance to herbicide, etc.

8. Production of Useful Biochemicals:
In some cases, plant cells in culture produce specialized biochemicals like alkaloids (nicotine, atropine, etc.), glycosides, steroids, and many other important substances.

9. Production of Transgenic Plant:
Desirable foreign genes introduced in plant cells can be cultured, and induced to multiply and differentiate to form plantlets. These plantlets when transferred to soil, grow into mature plants.

10. Embryo Rescue:
Embryo rescue is one of the earliest and most successful forms of in vitro culture techniques that are used to assist in the development of plant embryos that might not survive to become viable plants. Embryo rescue plays an important role in modern plant breeding, allowing the development of many interspecific and intergeneric food and ornamental plant crop hybrids. This technique nurtures the immature or weak embryo, thus allowing it to survive. Plant embryos are multicellular structures that have the potential to develop into a new plant. The most widely used embryo rescue procedure is referred to as embryo culture and involves excising plant embryos and placing them into media culture.

Embryo rescue is most often used to create interspecific and intergeneric crosses that would normally produce seeds that are aborted. Interspecific incompatibility in plants can occur for many reasons, but most often embryo abortion occurs. In plant breeding, wide hybridization crosses can result in small shrunken seeds which indicate that fertilization has occurred, however, the seed fails to develop. Many times, remote hybridizations will fail to undergo normal sexual reproduction, thus embryo rescue can assist in circumventing this problem.

There are the following stages of embryo rescue:

• Embryo Culture: The important parts of the embryo culture are The proper culture medium should be selected that supports the progressive and ordered development of the embryo, The bulbosum method of haploid production is the common excision methodology of the embryo.
• Ovule Culture: This is the technique for raising hybrids that normally fail to develop due to the abortion of the embryo during an early stage. Here, the addition of kinetin or fruit juice increases the initial growth.
• Ovary Culture: It is the most important technique for raising the interspecific hybrids between sexually incompatible species, e.g., in Anethum, the addition of kinetin in the medium, may result in the formation of polyembryony which give rise to multiple shoots.

Applications of Tissue Culture:

• Breeding of incompatible interspecific and intergeneric species.
• Overcoming seed dormancy.
• Determination of seed viability.
• Recovery of maternal haploids that develop as a result of chromosome elimination following interspecific hybridization.
• Used in studies on the physiology of seed germination and development.

Both plant and animal cells, tissues, and organ culture is possible in an artificial nutrient medium in controlled laboratory conditions. Many desired products can be obtained through tissue culture like pharmaceutical drugs, vaccines, monoclonal antibodies etc.

Differences between Callus Culture and Suspension Culture:

Features	Callus Culture	Suspension Culture
1. Nutrient Medium	Culture occurs on an agar solid medium.	Culture occurs in a liquid medium.
2. Cultured Ingredients	By cell division, a callus is formed.	A single cell or small group of cells is formed.
3. Time	2 – 3 weeks required.	5 – 10 days required.
4. Hormone	It contains auxin and cytokinin.	It contains only auxin like growth regulator.
5. Agitation	It needs no agitation.	It must be constantly agitated at 100 – 250 rpm.

Micropropagation

The rapid process of vegetative multiplication of plant material in tissue culture and their in vitro propagation technique is called micropropagation. Micropropagation is the practice of rapidly multiplying stock plant material to produce a large number of progeny plants, using modern plant tissue culture methods.

Micropropagation is used to multiply novel plants, such as those that have been genetically modified or bred through conventional plant breeding methods. It is also used to provide a sufficient number of plantlets for planting from a stock plant that does not produce seeds or does not respond well to vegetative reproduction.

Micropropagation can be achieved by the following methods:
1. Multiple Shootlet Production:
Shoot tips are used for tissue culture and raising mini plants. Shoot tips in culture medium produce multiple buds and each bud grows into a shoot. By using rooting hormone, the shoot is induced to produce roots. Micropropagation by this method is successfully employed in potatoes, cardamom, raspberry, peach, almond, orchids, bananas, etc.

2. Somatic Embryogenesis:
The embryo developed from a single somatic cell by tissue culture is known as a somatic embryo or embryoid. In carrots and alfalfa, the somatic embryos can be produced in thousands in a small volume of nutrient medium.

Stages of Micropropagation

Micropropagation usually comprises the following three stages (Murashige, 1974). The stages are

• Selection of parent plant and sterilization of explant: Healthy and desirable plants are selected. Multiple courses of 70% alcohol washes of explants (either meristematic tissue or totipotent cell) occur and finally, they are rinsed in sterilized water.
• Transfer of explant in culture medium: Sterilized explants are transferred to a nutrient medium (previously prepared) containing nutrients.
• Incubation: The cultures are kept in a culture room at a suitable temperature. From the non-meristematic explants, the cells are divided and the formation of an unorganized mass of tissue i.e., callus occurs.
• Shoot formation in culture medium: The callus tissue is transferred to the hormone medium and shoot formation occurs.
• Transfer of shoots in root initiating medium The meristematic shoot transferred to the hormone medium again. So the root initiation occurs to give birth to the plantlet.
• Transfer of plantlets to soil: Plantlet is transferred to sterilized soil for hardening in a greenhouse environment. This is called hardening. Later on, they are planted directly in the soil.

Objectives of Micropropagation:

• Production of virus-free stock.
• To multiply plants whose multiplication rate is very low.
• To produce progenies which are genetically identical to their parents.
• To obtain genetic variability.
• Recovery of distant hybrids.
• Germplasm conservation.
• Germplasm exchange.
• Genetic transformation – addition or deletion of the gene.

Advantages of Micropropagation

The micropropagation technique is preferred over the conventional asexual propagation method because of the following:

• This technique helps in the rapid multiplication of plant material required for agriculture, horticulture, and forestry.
• By this technique, a large number of off-springs or plantlets can be obtained every year.
• Genetic uniformity is easily maintained.
• Cloning can be done throughout the year in a very small space under controlled conditions.
• Resistance to diseases can be developed in many plant species.
• Offsprings can be obtained from sterile plants or free hybrids of extraordinary characteristics.
• Valuable germplasm can be stored for a long time.
• Only a small amount of tissue is needed for the regeneration of millions of plants.
• It provides a means for the international exchange of plant materials.
• Micropropagation has been successfully done in many trees of high economic value.
• Production of artificial seeds in Eucalyptus has been successfully achieved (Muralidharan & Mascarenhas, 1989).
• Genetic transformation and in vitro regeneration of conifers have been successfully performed (Gupta, 1989).

Limitations of Micropropagation

• Requirement of sophisticated facilities.
• High production cost.
• Requirement of skill in handling and maintenance.
• Somaclonal variations may arise during in vitro culture when a callus phase is involved.
• For many valuable species, suitable micropropagation techniques are not available (e.g., mango).
• Vitrification can be a problem in some species.

The production of new plants from a small piece of plant tissue (or cells) removed from the growing tips of a plant in a suitable growth medium (called culture solution) is called tissue culture. The growth medium (or culture solution) used for growing plant tissues is very important in this process because it contains various plant nutrients in the form of ‘jelly’ (called agar) and plant hormones which are necessary for plant growth. The process of tissue culture for producing new plants is carried out as follows:

• A small piece of plant tissue is taken from the growing point of the plant (tip of the plant) and placed on a sterile jelly that contains nutrients and plant hormones.
• The hormones make the cells in the plant tissue divide rapidly producing many cells which form a shapeless lump of mass called a ‘callus’.
• The callus is then transferred to another jelly containing suitable plant hormones which stimulate the callus to develop roots.
• The callus with developed roots is then put on yet another jelly containing different hormones which stimulate the development of shoots.
• The callus having roots and shoots separates into tiny plantlets. In this way, many tiny plantlets are produced from just a few original plant cells (or tissue).
• The plantlets thus produced are transplanted into pots or soil where they can grow to form mature plants.

The tissue culture technique is being used increasingly for the production of ornamental plants like orchids, dahlia, carnations, chrysanthemums, etc. The production of plants by the method of tissue culture is also known as micropropagation (due to the extremely small amount of plant material used).

(a) A small piece of plant tissue is taken from the growing point (tip) of a plant

(b) Plant tissue grows to form callus

(c) Callus develops roots and shoots

(d) Many plantlets are produced. These can be transplanted into pots or soil

Advantages of Tissue Culture

• Tissue culture is a very fast technique. Thousands of plantlets can be produced in a few weeks’ time from a small amount of plant tissue.
• The new plants produced by tissue culture are disease free.
• Tissue culture can grow plants around the year, irrespective of weather or season.
• Very little space is needed for developing new plants by tissue culture.

Do Organisms Create Exact Copies of Themselves in Asexual Reproduction

Asexual reproduction usually results in the production of genetically identical offspring, the only genetic variation arises as a result of occasional inaccuracies in DNA replication (or DNA copying) at the time of cell division. This will become clear from the following discussion.

The material which carries genetic information from the parents to the offspring is DNA-Deoxyribo Nucleic Acid (which is present in the form of chromosomes in the nuclei of all the cells). The basis of. asexual reproduction is mitosis. This is the division of a nucleus into two identical daughter nuclei (see Figure). Each daughter nucleus has the same genetic makeup because of the replication of DNA (or copying of DNA) of the parent cell. After the division of the nucleus, the rest of the parent cell divides to form two genetically identical daughter cells. The daughter cells can then form two offspring. From this, we conclude that all the offsprings produced by one parent as a result of asexual reproduction are usually genetically identical.

The new organisms (or offspring) produced by one parent through asexual reproduction (which are genetically identical to the parent) are called clones. The clones possess exact copies of the DNA (or genes) of their parent and hence show remarkable similarity to the parent and to one another. Thus, asexual reproduction produces genetically identical offspring called clones. For example, when a parent Hydra reproduces by the asexual method of budding, the new Hydrae (or offsprings) formed are clones (which are genetically identical to the parent Hydra as well as to one another).

Similarly, when we are using a cutting to grow a new plant, we are making a clone. The cutting of a plant contains the same DNA (or genes) as the original plant (or parent plant). This cutting will grow into an exact copy of the original plant. So, a clone is formed. The clones of plants can be produced by asexual methods of reproduction such as cuttings, layering, grafting, tissue culture, etc. These days techniques have been developed to clone even animals. Dolly the Sheep hit the headlines in 1997 as the first successfully produced animal clone. The process of producing genetically identical new organisms (or offsprings) by asexual reproduction methods is called cloning.

We will now explain how slight variations are introduced in asexual reproduction. The replication (or copying) of DNA in the cell is done by certain biochemical reactions which synthesize more of genetic material. No biochemical reaction can reproduce 100 percent same results. So, when the DNA already present in the nucleus of the parent cell is replicated (or copied) by making more DNA at the time of asexual reproduction, then slight variations come in the two copies formed. Due to this, the two DNA molecules formed by replication will be similar but may not be exactly identical to the original DNA. These slight variations in the replication of DNA molecules will lead to slight variations in the offspring produced by asexual reproduction.

From the above discussion, we conclude that the importance of DNA replication (or DNA copying) in asexual reproduction is that slight variations may arise in the offspring with respect to the parent organism. So, although the offspring produced by asexual reproduction are said to be genetically the same as the parent organism, still they have occasional variations. This means that the organisms do not always create exact copies of themselves in asexual reproduction. Please note that the importance of DNA copying in asexual reproduction is that the characteristics of the parent organisms are transmitted to its offspring and at the same time some occasional variations are also produced in the offspring. We will now describe the importance of variations introduced in reproduction.

The importance of variations in organisms introduced during reproduction is that it helps the species of various organisms to survive and flourish even in adverse environments. This will become clear from the following discussion. There may be some drastic changes like excessive heat or cold or shortage of water (drought), etc., in the habitat of a species of organisms. Now, if all the organisms of a population living in that habitat are exactly identical, then there is a danger that all of them may die and no one would survive under those conditions. This will eliminate the species from that habitat completely.

However, if some variations are present in some individual organisms to tolerate excessive heat or cold or survive on a meager water supply, then there is a chance for them to survive and flourish even in an adverse environment. In this way, the introduction of variations during reproduction provides stability to the populations of various species by preventing them from getting wiped out during adverse conditions. For example, if there is a population of certain bacteria living in temperate water (which is neither very hot nor very cold) and the temperature of water increases too much due to global warming, then most of these bacteria will not be able to tolerate excessive heat and hence die. But some bacteria which had variations to resist heat would survive and grow further.