Plant Tissue Culture

Chapter 26

Plant Tissue Culture

INTRODUCTION

• Tissue culture is in vitro cultivation of plant cell or tissue under aseptic and controlled environmental conditions, in liquid or on semisolid well-defined nutrient medium for the production of primary and secondary metabolites or to regenerate plant. This technique affords alternative solution to problems arising due to current rate of extinction and decimation of flora and ecosystem.

• The whole process requires a well-equipped culture laboratory and nutrient medium. This process involves various steps, viz. preparation of nutrient medium containing inorganic and organic salts, supplemented with vitamins, plant growth hormone(s) and amino acids as well as sterilization of explant (source of plant tissue), glassware and other accessories inoculation and incubation.

Advantages of Tissue Culture Technique over the Conventional Cultivation Techniques

Availability of raw material 

• Some plants are difficult to cultivate and are also not available in abundance. In such a case, the biochemicals/bioproducts from these plants cannot be obtained economically in sufficient quantity. Unlimited cutting of plants also leads to deforestation, natural imbalance and sometimes may lead to extinction of a particular species. Hence, tissue culture is considered a better source for regular and uniform supply of raw material, manageable under regulated and reproducible conditions in the medicinal plants industry for the production of phytopharmaceuticals.

Fluctuation in supplies and quality 

• The production of crude drugs is subject to variation in quality due to changes in climate, crop diseases and seasons. The method of collection, drying and storing also influence the quality of crude drug. All these problems can be overcome by tissue culture techniques.

Patent rights 

• Naturally occurring plants or their metabolites cannot be patented as such. Only a novel method of isolation can be patented. For R and D purpose, the industry has to spend a lot of money and time to launch a new natural product but can’t have patent right. Hence, industries prefer tissue culture for production of biochemical compounds. By this method, it is possible to obtain a constant supply and new methods can be developed for isolation and improvement of yield, which can be patented.

Political reasons 

• If a natural drug is successfully marketed in a particular country of its origin, the government may prohibit its export to up-value its own exports by supplying its phytochemical product, e.g. Rauwolfia serpentina and Dios Corea spp. from India. Similarly, the production of opium in the world is governed as such by political consideration, in such case, if work is going on the same drug; it will be either hindered or stopped. Here also, plant tissue culture is the solution.

Easy purification of the compound

• The natural products from plant tissue culture may be easily purified because of the absence of significant amounts of pigments and other unwanted impurities. With the advancement of modern technology in plant tissue culture, it is also possible to biosynthesize those chemical compounds which are difficult or impossible to synthesize.

Modifications in chemical structure

• Some specific compounds can be achieved more easily in cultured plant cells rather than by chemical synthesis or by microorganism.

Disease-free and desired propagule

• Plant tissue culture is advantageous over conventional method of propagation in large-scale production of diseasefree and desired propagules in limited space and also the germplasm could be stored and maintained without any damage during transportation for subsequent plantation.

Crop improvement 

• Plant tissue culture is advantageous over the conventional cultivation technique in crop improvement by somatic hybridization or by production of hybrids.

Biosynthetic pathway

•  Tissue culture can be used for tracing the biosynthetic pathways of secondary metabolites using labelled precursor in the culture medium.

Immobilization of cells 

• Tissue culture can also be used for plants preservation by immobilization of cell further facilitating transportation and biotransformation.

HISTORY

• Although the feasibility of aseptic culture of cells, tissues and organs on defined nutrient medium had been recognized at the beginning of the century, but it is only some few decades ago that modern developments in the cultivation of plants cell as a callus or as a suspension liquid culture actually came into existence. It is only in the last two decades that its implication has been realized and in particular pharmaceutical importance of this modern technique was appreciated. The principles of tissue culture were involved as early as 1838–1839 in cell theory advanced by Schleiden and Schwein. But according to noted biologist Gathered (1985), the discovery of tissue culture could be considered with the Henri-Louis Dudamel du Manceau’s (1756) pioneering experiment on wound healing in plants, demonstrated spontaneous callus formation on the decorticated region of the Elm plant. Further contribution to plant tissue culture could be attributed with the Haberland’s hypothesis (1902) that a cell is capable of autonomy and have potential for totipotency (the potential of cell to develop into an organism by regeneration is termed as totipotency by Morgan); hence, the isolated plant cell should be capable of cultivation on artificial medium.

• The development of multicellular or multiorganed body of a higher organism from a single cell (zygote) supports the totipotent behavior of a cell. But Haberblandt and coworker have tried to demonstrate the hypothesis but could not succeed. In 1904, another physiologist Hannig started research work, by taking embryogenic tissue instead of single cells for in vitro cultivation in an artificial medium consisting of mineral salts and sugar solution. He excised nearly matured embryos of some crucifers (Raphanus sativus, R. landra, R. caudates and Cochlearia Donica) and successfully cultivated them up to maturity. Thus, it became an important area of investigation, using an in vitro technique.

• Simon (1908) obtained more promising results as he achieved success in the regeneration of bulky callus, buds and roots from popular stem segments, and thus he succeeded in establishing the basis for callus culture and to some extent also micro-propagation.

• In vitro technique of culture was carried out further by many biologists. In 1922, Kotte (Germany) and Robbin (United States) simultaneously conceived a new approach to tissue culture and reported that true in vitro culture could be made easier by using meristematic cells (root tips or buds). Kotte carried out number of experiments and successfully cultivated small, excised root tips of pea, and grew the culture for two weeks by using a variety of nutrients containing salts of Knop’s solution, glucose and several nitrogenous compounds (such as asparagine, alanine and yeast extract). Robbin working independently maintained maize root tip culture for longer period by sub-culturing, but growth gradually diminished and ultimately culture was lost.

• White (1934–39) carried out the in vitro technique of tissue culture by changing the nature of media. He replaced the yeast extract in a medium containing inorganic salts and sucrose, with three vitamins (pyridoxine, thiamine and nicotinic acid) and was able to maintain the root tip culture; hence, White’s synthetic media later proved to be one of the basic media for cell and tissue culture.

• Gautheret (1934) successfully cultured cambium cells of some tree species (Acer pseudoplatanus, Ulmus campestre, Robinia pseudo acacia and Salix caprea) on the surface of the media (Knop’s solution containing glucose and cysteine hydrochloride) solidified with agar and observed that after six-month, proliferation of callus was ceased but on addition of auxin enhanced the proliferation of cambial culture and making it possible to prepare sub-culture.

• Van Overbeek et al. (1941) used coconut milk (embryo sac fluid) for embryo development and callus formation in Datura, which proved to be turning point in the development of embryo culture, which latter on proved to be helpful in the development of several hybrids.

• Loo (1945) got success in developing whole plant from stem tip culture. He obtained excellent cultures from stem tips of Dodder and Asparagus. Subsequently, Ball (1946) was able to identify the exact part of the shoot meristem, which gave rise to whole plant. This method is now being used in plant propagation at industrial scale throughout the world.

• Muir (1953) demonstrated that on transferring the callus tissues of these two plants into liquid medium and on subsequent agitating on a shaking machine, it is possible to break down the callus tissue into single cell and small cell aggregates, which on sub-culturing into fresh liquid medium can multiply while remaining in the medium under constant shaking. Muir and associates (1954) reported that the pieces of callus of Tagetes erect and Nicotiana tabacum can be cultured in the form of cell suspension.

• Van Overbeek et al. (1941) had suggested earlier that liquid endosperm (coconut milk) is a good medium for embryo culture. Later in 1955, Skoog and coworker finally isolated adenine derivative from the embryo sac known as kinetin which helps in the proliferation of embryo.

• Skoog and Miller (1957) proposed the concept of hormonal control of organ formation. They demonstrated that root and bud initiation were conditioned by balance between auxin and kinetin addition to other ingredients of the define medium. High proportions of auxin promoted rooting, whereas proportionately more kinetin-initiated bud or shoot formation.

• Bergmann (1960) developed plating technique for cloning a large number of isolated single cells. He demonstrated the technique by using callus culture of Nicotiana tabacum and Phaseolus vulgaris and reported population of nearly 90% of free cells. In the same year, i.e. 1960, Jones et al. used to hang drops of free cells for the micro-culture propagation. This technique proved useful to have continuous observation of cell growth in the culture.

• In 1960, Cocking introduced protoplasmic plant tissue culture. He succeeded in isolating the protoplasts of plant tissue by using cell wall enzymes like cellulase, hemicellulase, pectinase and protease. The enzyme was extracted from fungus Trichoderma viride. Earlier, Michel (1939) had demonstrated the role of sodium nitrate fusion of protoplasts. In the same year, Steward and coworker had successfully raised a large number of plantlets from carrot root suspension culture. In year 1960, Moral initiated micropropagation technique and produced virus-free orchid, Cymbidium.

• Steward and coworker in 1966 raised large number of plantlets from carrot root suspension culture via somatic embryogenesis. Actually Rienert (1968) introduced somatic embryogenesis callus, cultured on a semisolid medium. This phenomenon of somatic embryogenesis for the production of plantlets was later reported in many species. All these discoveries contributed to the establishment of totipotency power of the cells under suitable environment thereby accomplishing theory introduced by Haberblandt. 

• In 1970, Power et al. demonstrated the intra- and interspecific fusion between the protoplasts of different plant roots; subsequently, in 1972, Carlson et al., succeeded in obtaining the first inter-specific somatic hybrid by protoplasts fusion of Nicotiana species (N. glauca and N. longsdorfi). In 1981, Vilnken brought new approach of electrical fusion of protoplasts. Later Gamborg and Neabors (1987) described a number of variations in protoplasts fusion.

BASIC REQUIREMENTS FOR A TISSUE CULTURE LABORATORY

• For the successful achievement of any type of tissue culture technique, a tissue culture laboratory should have the following general basic facilities:

1. Equipment and apparatus 
2. Washing and storage facilities 
3. Media preparation room 
4. Sterilization room 
5. Aseptic chamber for culture 
6. Culture rooms or incubators fully equipped with temperature, light and humidity control devices. 
7.  Observation or recording area well equipped with computer for data processing.

Equipment and Apparatus

Culture vessels and glassware 

• Many different kinds of vessels may be used for wing cultures. Callus culture can be grown successfully in large test tubes (25 × 150 mm) or wide mouth conical flasks (Erlenmeyer flask). In addition to the culture vessels, glassware such as graduated pipettes, measuring cylinders, beakers, filters, funnel and petri dishes are also required for making preparations. All the glass wares should be of Pyrex or corning.

Equipment 

• Scissors, scalpels and forceps for explant preparation from excised plant parts and for their transfer

1. A spirit burner or gas micro-burner for flame sterilization of instruments 
2. An autoclave to sterilize the media 
3. Hot air oven for the sterilization of glassware, etc. 
4. A pH meter for adjusting the pH of the medium 
5. A shaker to maintain cell suspension culture 
6. A balance to weigh various nutrients for the preparation of the medium 
7. Incubating chamber or laminar airflow with UV light fitting for aseptic transfer of explants to the medium and for sub-culturing 
8. A BOD incubator for maintaining constant temperature to facilitate the culture of callus and its subsequent maintenance

Washing and storage facilities

• First and foremost requirement of the tissue culture laboratory is provision for fresh water supply and disposal of the waste water, and space for distillation unit for the supply of distilled and double distilled water and de-ionized water. Acid and alkali resistant sink or wash basin for apparatus/ equipment washing and the working table should also be acid- and alkali-resistant.

• Sufficient space is required for placing hot air oven, washing machine, pipette washers and the plastic bucket or steel tray for soaking or drainage of the detergent bath or extra water. For the storage of dried glassware separate dust proof cupboards or cabinet should be provided. It is mandatory to maintain cleanliness in the area of washing, drying and storage.

Media preparation room 

• Media preparation room should have sufficient space to accommodate chemicals, lab ware, culture vessels and equipments required for weighing and mixing, hot plate, pH meter, water baths, Bunsen burners with gas supply, microwave oven, autoclave or domestic pressure cooker, refrigerator and freezer for storage of prepared media and stock solutions.

Sterilization Room 

• For the sterilization of culture media, a good quality ISI mark autoclave is required and for small amount domestic pressure cookers, can also serve the purpose. For the sterilization of glassware and metallic equipments hot air oven with adjustable tray is required.

Aseptic chamber/area for transfer of culture 

• For the transfer of culture into sterilized media, contaminant-free environment is mandatory. The simplest type of transfer area requires an ordinary type of small wooden hood, having a glass or plastic door either sliding or hinged fitted with UV tube. This aseptic hood can be conveniently placed in a quiet corner of the laboratory. These days, modern laboratory have laminar airflow cabinet having vertical or horizontal airflow, arrange over the working surface to make it free from dust particles/ micro-contaminants.

• The air coming out of the fine filter (a 0.3-μm HEPA filter) is ultra-clean (free from fungal or bacterial contaminant) and having adequate velocity (27±3 m/min) to prevent micro-contamination of the working area by worker sitting in front of the cabinet.

• Inside the cabinet, there is arrangement for Bunsen burner and a UV tube fitted on the ceiling of the cabinet (to make area free from any live contamination). The advantage of working in the laminar airflow cabinet is that the flow of air does not hamper the use of Bunsen burner and moreover, the cabinet occupies relatively small space within the laboratory 

Incubation room or incubator 

• Environmental factors have great effect on the growth and differentiation of cultured tissues. Therefore, it is very much essential to incubate all types of cultures in well-controlled environmental conditions, like temperature, humidity, illumination and air circulation. A typical incubation chamber or area should have both light and temperature-controlled devices managed for 24 h period. Air conditioners or room heaters are required to maintain the temperature at 25±2°C. Light is adjusted in the terms of photo period duration (specified period for total darkness as well as for higher intensity light). Further the requirement for humidity range of 20–90% controllable to ±3% and uniform forced air circulation can be achieved.

• The incubation chamber or room should have the provision for storing the culture vessels (flask, jars and petri dishes). Shelves should be designed in such a way so that the culture vessels can be placed in the shelf or trays in such a way that there should not be any hindrance in the light, temperature and humidity maintenance. A label having full detail about date of inoculation, name of the explant, medium and any other special information should be stuck on each tray and rack to ensure identity and for maintaining the data of experiment. In the case of suspension culture arrangement for shaker should also be made.

• These days BOD incubators with all the requisite environmental condition maintenance are available in the market, they occupy less space and manageable with small generator or automatic invertor in the case of electricity failure to maintain the necessary light and temperature conditions. Failure of electricity may spoil important experiment and in the case of suspension culture the whole culture may get damaged due to stoppage of the shaker.

• BOD incubators required to maintain the culture conditions should have the following characteristics:

1. Temperature range, 2–40°C 
2. Temperature control ±0.5°C
3. Automatic digital temperature recorder 
4. 24-h temperature and light programming 
5. Adjustable fluorescent lighting up to 10,000 lux 
6. Relative humidity range 20–98% 
7. Relative humidity control ±3% 
8. Uniform forced air circulation 
9. Shaker
10. Capacity up to 0.7 m3 of 0.5 m2 shelf space

Data collection and recording the observation 

• The growth and maintenance of the tissue culture in the incubator should be observed and recorded at regular intervals. All the observations should be done in aseptic environment, i.e., in the laminar airflow. Whereas for microscopic examination, separate dust-free space should be marked for microscopic work. All the recorded data should be fed into the computer.

GENERAL PROCEDURES INVOLVED IN PLANT TISSUE CULTURE

• In vitro culturing of plant tissue involves the following steps:

1. Sterilization of glassware tools/vessels 
2. Preparation and sterilization of explant 
3. Production of callus from explant 
4. Proliferation of cultured callus  
5. Sub-culturing of callus 
6. Suspension culture

Sterilization of Glassware tools/vessels

 Cleaning of glassware 

• All the glassware to be used in tissue culture laboratory should be of Pyrex or corning. To make them free from any dirt, waxy material or bacteria, all the glassware should.

• be kept overnight dipped in sodium dichromate-sulphonic acid solution. Next morning, glassware should be washed with fresh running tap water, followed by distilled water and placed in inverted position in plastic bucket or trays to remove the extra water. For drying the glassware, it is placed in hot air oven at high temperature about 120°C for 1/2–1 h

• In the case of plastic labware, washing should be carried out with a mild nonabrasive detergent followed by washing under tap water or the plasticware after general washing with dilute sodium bicarbonate and water followed by drainage of extra water, rinsed with an organic solvent such as alcohol, acetone and chloroform. Washed and dried glassware or plasticware should be stored in dust proof cupboards.

• To prevent reinfection following sterilization, empty containers are wrapped with aluminum foil. Stainless steel, metal tools (knives, scalpels, forceps, etc.) are also wrapped with the aluminum foil and pads of cotton wool are stuffed into the opening of the pipettes, which are either also wrapped in aluminum or placed in an aluminum or stainless-steel box. The period of sterilization usually ranges between 1 and 4 h.

Preparation of Explant

• Explant can be defined as a portion of plant body, which has been taken from the plant to establish a culture. Explant can be obtained from plants, which are grown in controlled environmental conditions. Such plants will be usually free from pathogens and are homozygous in nature. Explant may be taken from any part of the plant like root, stem, leaf, or meristematic tissue like cambium, floral parts like anthers, stamens, etc.

• Age of the explant is also an important factor in callus production. Young tissues are more suitable than mature tissues. A suitable portion from the plant is removed with the help of sharp knife, and the dried and mature portions are separated from young tissue. When seeds and grains are used for explant preparation, they are directly sterilized and put in nutrient medium. After germination, the obtained seedlings are to be used for explant preparation.

Surface Sterilization of Explant

• For the surface sterilization of the explant, chromic acid, mercuric chloride (0.11%), calcium hypochlorite, sodium hypochlorite (1–2%) and alcohol (70%) are used. Usually the tissue is immersed in the solution of sterilizing agent for 10 s to 15 min, and then they are washed with distilled water. Repeat the treatment with sodium hypochlorite for 20 min, and the tissue is finally washed with sterile water to remove sodium hypochlorite. Such tissue is used for inoculation

• The explants are sterilized by exposing to aqueous sterilized solution of different concentration as shown in Table 26.1. In the case of leaf or green fresh stem the explant needs pretreatment with wetting agent (70–90% ethyl alcohol, Tween 20), 5–20 drops in 100 ml of purified water or some other mild detergent to be added directly into the sterilization solution to reduce the water repulsion (due to waxy secretion).

• Procedure to be followed for respective explant is as follow:

Seeds

• 1st Step: Dip the seeds into absolute ethyl alcohol for 10 s and rinse with purified water.
• 2nd Step: Expose seeds for 20–30 min to 10% w/v aqueous calcium hypochlorite or for 5 min in a 1% solution of bromine water.
• 3rd Step: Wash the treated seeds with sterile water (three to five times) followed by germination on damp sterile filter paper.

Fruits

• 1st Step: Rinse the fruit with absolute alcohol. 

• 2nd Step: Submerge into 2% (w/v) solution sodium hypochlorite for 10 min. 

• 3rd Step: Washing repeated with sterile water and remove seeds of interior tissue.

Stem

• 1st Step: Clean the explant with running tap water followed by rinsing with pure alcohol. 

• 2nd Step: Submerge in 2% (w/v) sodium hypochlorite solution for 15–30 min. 

• 3rd Step: Wash three times with sterile water.

Leaves

• Clean the leaf explant with purified water to make it free from dirt and rub the surface with absolute ethyl alcohol. Dip the explant in 0.1% (w/v) mercuric chloride solution, wash with sterile water to make it free from chloride and then dry the surface with sterile tissue paper.

Production of Callus from Explant

• The sterilized explant is transferred aseptically onto defined medium contained in flasks. The flasks are transferred to BOD incubator for maintenance of culture. Temperature is adjusted to 25±2ºC. Some amount of light is necessary for callus (undifferentiated amorphous cell mass) production. Usually, sufficient amount of callus is produced within three to eight days of incubation.

Proliferation of Callus

• If callus is well developed, it should be cut into small pieces and transferred to another fresh medium containing an altered composition of hormones, which supports growth. The medium used for production of more amount of callus is called proliferation medium.

Sub-culturing of Callus

• After sufficient growth of callus, it should be periodically transferred to fresh medium to maintain the viability of cells. This sub-culturing will be done at an interval of 4–6 weeks.

Suspension Culture

• Suspension culture contains a uniform suspension of separate cells in liquid medium. For the preparation of suspension culture, callus is transferred to liquid medium, which is agitated continuously to keep the cells separate. Agitation can be achieved by rotary shaker system attached within the incubator at a rate of 50–150 rpm. After the production of sufficient number of cells sub-culturing can be done.

CULTURE MEDIA

• Nutritional requirements for optimal growth of a tissue culture may vary with the species. Even tissues from different parts of a plant may have different requirements for proper satisfactory growth. As such no single medium can be suggested as being entirely sufficient for the satisfactory growth of all types of plant tissues and organs; hence, with every new system it is essential to work out a medium by hit and trial that would fulfil the specific requirements of that particular tissue. List of several culture media developed by scientists to culture diverse tissues and organs are Gauthe ret (1942), White (1943), Haberland et al. (1946), Haller (1953), Nitsch and Nitsch (1956), Murashige and Skoog (1962), Eriksson (1965) and B5 (Gamberg et al., 1968) 

Media Composition

• To maintain the vital functions of a culture, the basic medium consisting of inorganic nutrients (macronutrients and micronutrients) adapted to the requirements of the object in question, must be supplemented with organic components (amino acids, vitamins), growth regulators (phytohormones) and utilizable carbon (sugar) source and a gelling agent (agar/phytagel).

Inorganic nutrients

• Mineral elements play very important role in the growth of a plant. For example, magnesium is a part of chlorophyll molecule, calcium is a component of cell wall and nitrogen is an important element of amino acids, vitamins, proteins and nucleic acids. Iron, zinc and molybdenum are parts of certain enzymes. Essentially about 15 elements found important for whole plant growth have also been proved necessary for the growth of tissue(s) in culture.

• Macronutrients: The macronutrients include six major elements: nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg) and Sulphur (S) present as salts that constitute the various above mentioned defined media. The concentration of the major elements like calcium, phosphorus, Sulphur and magnesium should be in the range of 1–3 mmol l-1 whereas the nitrogen in the media (contributed by both nitrate and ammonia) should be 2–20 mmol l-1 

• Micronutrients: The inorganic elements required in small quantities but essential for proper growth of plant cells or tissues are boron (B), copper (Cu), iron (Fe), manganese (Mn), zinc (Zn) and molybdenum (Mo). Out of these, iron seems more critical as it is used in chelated forms of iron and zinc in preparing the culture media, as iron tartrate and citrate are difficult to dissolve. The concentration generally prescribed for all these elements are in traces.

• These are added to culture media depending upon the requirement of the objective. In addition to these elements, certain media are also enriched with cobalt (Co), iodine (I) and sodium (Na) but exact cell growth requirement is not well established.

• The composition of some plant tissue culture media reveals that the chief difference in the composition of various commonly used tissue culture media lies in the quantity of various salts and ions. Qualitatively, the inorganic nutrients required for various culture media appear to be fairly constant. The active factor in the medium is the ions of different types rather than the salt (mineral salts on dissolving in water undergo dissociation and ionization). A single ion may be contributed by more than one salt. For example, in Murashige and Skoog’s medium, NO3 - ions are contributed by NH4 NO3 as well as KNO3 and K+ ions are contributed by KNO3 and KH2 PO4.

• White’s medium, one of the earliest plant tissue culture media, includes all the necessary nutrients and was widely used for root culture. The experience of various investigators has however revealed that quantitatively the inorganic nutrients are inadequate for good callus growth (Murashige and Skoog’s, 1962); hence, most plant tissue culture media that are now being widely used are richer in mineral salts (ions) as compared to White’s medium. Aluminum and nickel used by Heller’s (1953) could not be proved to be essential and, therefore, were dropped by subsequent workers, but sodium, chloride and iodide are indispensable.

• In Heller medium, special emphasis was given to iron and nitrogen. In the original White’s medium iron was used in the form of Fe2 (SO4), but Street and coworkers replaced it by FeCl3 for root culture because of the impurities due to Mn and some other metallic ions. However, FeCl3 also did not prove to be an entirely satisfactory source of iron. In this form iron is available to the tissue culture at or around pH 5.2 and within a week of inoculation the pH of the medium drift from 4.9–5.0 to 5.8–6.0, and the root culture started showing the iron deficiency symptoms. To overcome this difficulty, in most medium, iron is now used as FeEDTA; in this form, iron remains available up to a pH of 7.6–8.0. However, unlike root, callus cultures can utilize FeCl3 to pH 6.0 by secreting natural chelates. FeEDTA may be prepared by using Fe2 (SO4 )3 7H2 O and Na2 EDTA 2H2 O.

Organic nutrients

• Nitrogenous substances: Most cultured plant cells are capable of synthesizing essential vitamins but not in sufficient amount. To achieve best growth, it is essential to supplement the tissue culture medium with one or more vitamins and amino acid. Among the essential vitamins thiamine (vitamin B1) has been proved to be essential ingredient. Other vitamins, especially pyridoxine (vitamin B6), nicotinic acid (vitamin B3) and calcium pantothenate (vitamin B5) and inositol are also known to improve growth of the tissue culture material. As shown in Table 26.2, there is variation in the quantities of essential vitamins used by various standard media.

• Numerous complex nutritive mixtures of undefined composition, like casein hydrolysate, coconut milk, corn milk, malt extract, tomato juice and yeast extract have also been used to promote growth of the tissue culture, but these substances specifically fruit extracts may affect the reproducibility of results because of variation in the quality and quantity of growth promoting constituent in these extracts.

• Carbon Source: It is essential to supplement the tissue culture media with an utilizable source of carbon to the culture media. Haberland (1902) attempted to culture green mesophyll cells, probably with the idea that green cells would have simple nutritive requirement, but this did not prove to be true. In fact even fully organized green shoot in cultures, and it also did not show proper growth and proliferation without the addition of suitable carbon source in the medium.

• The most commonly used carbon source is sucrose at a concentration of 2–5%. Glucose and fructose are also known to be used for good growth of some tissues. Ball (1953, 1955) demonstrated that autoclaved sucrose was better than filtered sterilized sucrose. Autoclaving may do the hydrolysis of the sucrose thereby converting it into more efficiently utilizable sugar such as fructose. In general, excised dicotyledonous roots grow better with sucrose whereas monocots do best with dextrose (glucose). Some other forms of carbon that plant tissues are known to utilize include maltose, galactose, mannose, lactose and sorbitol. It has been reported that some tissues can even metabolize starch as the sole carbon source, e.g., tissue cultures of sequoia and maize endosperm.

Plant growth regulators

• Plant growth regulators are the critical media components in determining the developmental pathway of the plant cells. The plant growth regulators used most commonly are plant hormones or their synthetic analogues.

Classes of plant growth regulators:

• There are five main classes of plant growth regulator used in plant cell culture, namely: 

1. auxins, 
2. cytokinin's,
3. gibberellins, 
4. abscisic acid and 
5. ethylene

Auxins: 

• Auxins promote both cell division and cell growth. The most important naturally occurring auxin is IAA (indole-3-acetic acid), but its use in plant cell culture media is limited because it is unstable to both heat and light. Occasionally, amino acid conjugates of IAA (such as indole– acetyl–L-alanine and indole–acetyl–L-glycine), which are more stable, are used to partially alleviate the problems associated with the use of IAA. It is more common, though, to use stable chemical analogues of IAA as a source of auxin in plant cell culture media. 2,4-Dichlorophenoxyacetic acid (2,4-D) is the most commonly used auxin and is extremely effective in most circumstances. Other auxins are available, and some may be more effective or ‘potent’ than 2,4-D in some instances.

Cytokinins: 

• Cytokinin promote cell division. Naturally occurring cytokinins are a large group of structurally related (they are purine derivatives) compounds. Of the naturally occurring cytokinins, two have some use in plant tissue culture media (Table 26.4). These are zeatin and 2iP (2-isopentyl adenine). Their use is not widespread as they are expensive (particularly zeatin) and relatively unstable. The synthetic analogues, kinetin and BAP (benzyl aminopurine) are therefore used more frequently. Nonpurine based chemicals, such as substituted hexylureas, are also used as cytokinins in plant cell culture media. These substituted hexylureas can also substitute for auxin in some culture systems.

Gibberellins: 

• There are numerous, naturally occurring, structurally related compounds termed gibberellins. They are involved in regulating cell elongation and are agronomically important in determining plant height and fruit set. Only a few of the gibberellins are used in plant tissue culture media, GA3 being the most common.

Abscisic acid:

•  Abscisic acid (ABA) inhibits cell division. It is most commonly used in plant tissue culture to promote distinct developmental pathways such as somatic embryogenesis.

Ethylene: 

• Ethylene is a gaseous, naturally occurring, plant growth regulator most commonly associated with controlling fruit ripening in climacteric fruits, and its use in plant tissue culture is not widespread. It does, though, present a particular problem for plant tissue culture. Some plant cell cultures produce ethylene, which, if it builds up sufficiently, can inhibit the growth and development of the culture. The type of culture vessel used, and its means of closure affect the gaseous exchange between the culture vessel and the outside atmosphere and thus the levels of ethylene present in the culture.

Solidifying agents for solidification of the media 

• Due to improved oxygen supply and support to the culture growth, solid media are often preferred to liquid cultures. For this purpose, substance with strong gelling capacity is added into the liquid media. These reversibly bind water and thus ensure the humidity of the medium desired for culturing depending on the concentration.

Gelling agent used to solidify liquid media 

• The most commonly used substance for this purpose is the pyrocollodion agar–agar obtained from red algae (Gonidium Garcialara). It is generally used at a concentration of 0.8–1.0%, with higher concentration medium becoming hard and does not allow the diffusion of nutrients into the tissues medium. However, agar is not an essential component of the nutrient medium. Single cell and cell aggregates can be grown as suspension cultures in liquid medium containing inorganic, organic nutrients and other growth factors. Such culture should however be regularly aerated either by bubbling sterile air or gentle agitation. In nutritional studies, the use of agar should be avoided because of the impurities present in all the commercially available agar–agar especially of Ca, Mg, K, Na and trace elements.

• Agar (Agarose) is extraordinary resistant to enzymatic hydrolysis at incubation temperature, and only a few bacteria exist which are capable of producing degrading enzyme— agarase. This resistance to hydrolysis is the fundamental importance to the use of agar–agar in cell culture medium. It is also neutral to media constituents and thus do not react with them.

• Agar (Agarose) is extraordinary resistant to enzymatic hydrolysis at incubation temperature, and only a few bacteria exist which are capable of producing degrading enzyme— agarase. This resistance to hydrolysis is the fundamental importance to the use of agar–agar in cell culture medium. It is also neutral to media constituents and thus do not react with them.

Media Preparation

• For media preparation, there are two possible methods, 

1. To weigh the required quantity of nutrient, dissolve them separately and mix at the time of medium preparation.
2. To prepare the stock solution separately for macro-nutrients, micro-nutrients, iron solution and organic components are stored in the refrigerator till not used, e.g., Murashinge and Skoog’s media stock solution is prepared as is shown in the table.

Procedure

• All the ingredients may be grouped into following four groups:

Concentration of the ingredients 

• For the preparation of stock solution, the Group I ingredient are prepared at 20x concentrated solution, Group II at 200x, Group III Iron salts at 200x and Group IV organic ingredient except sucrose at 200x.

Solution preparation 

• For the preparation of stock solution, each component (analar grade) should be weighed and dissolved separately in glass distilled or demineralized water and then mixed together. Stock solution may be prepared at the strength of 1 mmol l-1 or 10 mmol l-1. All the stock solutions are stored in refrigerator till used.

• For iron solution, dissolve FeSO4 7H2 O and Na2 EDTA 2H2 O separately in about 450 ml distilled water by heating and constant stirring. Mix the two solutions, adjust pH of the medium to 5.5 and final volume adjusted to 1 L with distilled water.

Semisolid media preparation 

• Required quantities of agar and sucrose are weighed and dissolved in wafer by 3/4th volume of medium, by heating them on water bath. Adequate quantities of stock solution (for 1L medium 50 ml of stock solution of Group 1, 5 ml of stock solution II, III and IV group) and other special supplements are added and final volume is made up with double distilled water. After mixing well, pH of the medium is adjusted to 5.8 using 0.1 N NaOH and 0.1 N HCl.

Sterilization of Culture 

• Media Culture media packed in glass containers or vessels are sealed with cotton plugs and covered with aluminium foils and are autoclaved at pressure of 2–2.2 atm at 121°C for 15–40 min (time to be fixed from the time when temperature reaches the required temperature). The exposure time depends on the volume of the liquid to be sterilized.

• Minimum autoclaving time includes the time required for the liquid volume to reach the sterilizing temperature (121°C) and 15 min at this temperature. Time may vary due to difference in autoclaves. Moreover, the actual success of sterilization can be tested using a bio-indicator, commonly spores of the bacterium Bacillus stearothermophillus are used as such as a test organism. Together with culture medium and a pH indicator in ampoules sealed by melting, both autoclaved material and nonautoclaved controls are incubated for 24–48 h at 60°C. If the spores are dead, the colour of the pH indicator in the solution remains unchanged indicating no change in pH.

TYPES OF PLANT TISSUE CULTURES

• Plant tissue culture is a general term to culture the isolated plant organs (particularly of isolated roots but, to a lesser extent of stem tips, immature embryo, leaf primordia, flower structures and even the cells and the protoplasts) under aseptic environment.

Root Tip Culture

• Tips of the lateral roots are sterilized, excised and transferred to fresh medium. The lateral roots continue to grow and provide several roots, which after seven days, are used to initiate stock or experimental cultures. Thus, the root material derived from a single radicle could be multiplied and maintained in continuous culture; such genetically uniform root cultures are referred to as a clone of isolated roots.

Leaves or Leaf Primordia Culture

• Leaves (800 μm) may be detached from shoots, surface sterilized and placed on a solidified medium where they will remain in a healthy condition for a long period. Growth rate in culture depends on their stage of maturity at excision. Young leaves have more growth potential than the nearly mature ones.

Shoot Tip Culture

• The excised shoot tips (100–1000 μm long) of many plant species can be cultured on relatively simple nutrient media containing growth hormones and will often form roots and develop into whole plants.

Complete Flower Culture

• Nitsch in 1951 reported the successful culture of the flowers of several dicotyledonous species; the flowers remain healthy and develop normally to produce mature fruits. Flowers (2 days after pollination) are excised, sterilized by immersion in 5% calcium hypochlorite, washed with sterilized water and transferred to culture tubes containing an agar medium. Often fruits that develop are smaller than their natural counterpart, but the size can be increased by supplementing the medium with an appropriate combination of growth hormones.

Anther and Pollens Culture

• Young flower buds are removed from the plant and surface sterilized. The anthers are then carefully excised and transferred to an appropriate nutrient medium. Immature stage usually grows abnormally and there is no development of pollen grains from pollen mother cells. Anther at a very young stage (containing microspore mother cells or tetrads) and late stage (containing binucleate starch-filled pollen) of development are generally ineffective, and hence, for better response always select mature anther or pollen.

• Mature anther or pollen grains (microspore) of several species of gymnosperms can be induced to form callus by spreading them out on the surface of a suitable agar media. Mature pollen grains of angiosperms do not usually form callus, although there are one or two exceptions.

Ovule and Embryo Culture

• Embryo is dissected from the ovule and put into culture media. Very small globular embryos require a delicate balance of the hormones. Hence, mature embryos are excised from ripened seeds and cultured mainly to avoid inhibition in the seed for germination. This type of culture is relatively easy as the embryos require a simple nutrient medium containing mineral salts, sugar and agar for growth and development.

• The seeds are treated with 70% alcohol for about 2 min, washed with sterile distilled water, treated with surface sterilizing agent for specific period, once again rinsed with sterilized distilled water and kept for germination by placing them on double layers of presterilized filter paper placed in Petri dish moistened with sterilized distilled water or placed on moistened cotton swab in Petri dish. The seeds are germinated in dark at 25–28°C and small part of the seedling is utilized for the initiation of callus.

• Apart from above-mentioned cultures, there are two more methods for culturing of plant tissues/cells:

1. Protoplast culture and 
2. Hairy roots culture.

Protoplast Culture

• Protoplasts are the naked cells of varied origin without cell walls, which are cultivated in liquid as well as on solid media. Protoplasts can be isolated by mechanical or enzymatic method from almost all parts of the plant: roots, tubers, root nodules, leaves, fruits, endosperms, crown gall tissues, pollen mother cells and the cells of the callus tissue but the most appropriate is the leaves of the plant. 

• Fully expanded young leaves from the healthy plant are collected, washed with running tap water and sterilized by dipping in 70% ethanol for about a minute and then treated with 2% solution of sodium hypochlorite for 20–30 min, and washed with sterile distilled water to make it free from the trace of sodium hypochlorite.

• The lower surface of the sterilized leaf is peeled off and stripped leaves are cut into pieces (midrib). The peeled leaf segments are treated with enzymes (macerozyme and then treated with cellulase) to isolate the protoplasts.

• The protoplasts so obtained are cleaned by centrifugation and decantation method. Finally, the protoplast solution of known density (1 × 105 protoplasts/ml) is poured on sterile and cooled down molten nutrient medium in Petri dishes. Mix the two gently but quickly by rotating each Petri dish. Allow the medium to set and seal Petri dishes with paraffin film. Incubate the Petri dishes in inverted position in BOD incubator. The protoplasts, which are capable of dividing undergo cell divisions and form callus within 2–3 weeks. The callus is then sub-cultured on fresh medium. Embryogenesis begins from callus when it is transferred to a medium containing proper proportion of auxin and cytokinin, where the embryos develop into plantlets which may be transferred to pots.

Hairy Root Culture

• Steward et al. (1900). A large number of small fine hairy roots covered with root; hairs originate directly from the explant in response to Agrobacterium Rhizogen's infection are termed hairy roots. These are fast-growing, highly branched adventitious roots at the site of infection and can grow even on a hormone-free culture medium. Many plant cell culture systems, which did not produce adequate number of desired compounds, are being reinvestigated using hairy root culture methods. A diversified range of plant species has been transformed using various bacterial strains. One of the most important characteristics of the transformed roots is their capability to synthesize secondary metabolites specific to that plant species from which they have been developed. Growth kinetics and secondary metabolite production by hairy roots is highly stable and are of equal level and even they are higher to those of field grown plants.

ESTABLISHMENT AND MAINTENANCE OF VARIOUS CULTURES

• For the growth establishment and maintenance of various types of plant tissue cultures, there are three main culture systems, selected on the basis of the objective.

1. Growth of callus masses on solidified media (callus culture also known as static culture).  
2. Growth in liquid media (suspension culture) consists of mixture of single cells or cell aggregates. 
3.  Protoplast culture:

• Callus culture (static tissue culture) 
•  Suspension culture

Callus Culture

• Callus is an amorphous aggregate of loosely arranged parenchyma cells, which proliferate from mother cells. Cultivation of callus usually on a solidified nutrient medium under aseptic conditions is known as callus culture; unlike tumor tissue, the cell division takes place periclinally

Initiation of callus culture

Selection and preparation of explant 

• Selection: For the preparation of callus culture, organ or culture is selected such as segments of root or stem, leaf primordia, flower structure or fruit, etc

Preparation:

• Excised parts of the plant organ are first washed with tap water, and then sterilized with 0.1% of mercuric chloride (HgCl2 ) or 2% w/v, sodium hypochlorite (NaOCl) solution for 15 min. In the case of plant organ containing waxy layer, the material is either pretreated with wetting agents [ethanol 70–90%; tween 20 (polyoxyethylene sorbitan monolaurate): 1–20 drops into 100 ml distilled water]; or other detergents are added to the sterilization solution to reduce the water repulsion. 

• Wash the sterilized explants with sterile glass distilled water and cut aseptically into small segments (2–5 mm).

Selection of culture medium

The organ is to be cultured in well-defined nutrient medium containing inorganic and organic nutrients and vitamins. The culture of the medium depends on the species of the plant and the objective of the experiment. The MS medium is quite suitable for dicot tissues because of relatively high concentration of nitrate, potassium and ammonium ions in comparison to other media

Growth hormones (auxin, cytokinin) are adjusted in the medium according to the objective of the culture. For example, auxins, 1BA and NAA are widely used in medium for rooting and in combination with cytokinin for shoot proliferation. 2, 4-D and 2, 4, 5-T are effective for good growth of the callus culture. This is also quite favourable for monocot tissues or explant

The selected semisolid nutrient is prepared. The pH of the medium is adjusted (5.0–6.0) and poured into culture vessels (15 ml for 25 x 150 mm culture tubes or 50 for 150 ml flasks) plugged and sterilized by autoclaving.

Transfer of explant

Surface sterilized organs (explant) from stem, root or tuber or leaf, etc., are transferred aseptically into the vessel containing semisolid culture medium

Incubation of culture

The inoculated vessels are transferred into BOD incubator with auto controlled device. Incubate at 25–28°C using light and dark cycles for 12-h duration. Nutrient medium is supplemented with auxin to induce cell division. After three

to four weeks, callus should be about five times the size of the explant. Many tissue explants possess some degree of polarity with the result that the callus is formed most early at one surface. In stem segment, callus is formed particularly from that surface which in vivo is directed towards the root

The unique feature of callus is its ability to develop normal root and shoot, ultimately forming a plant. Commercially important secondary metabolites can also be obtained from static culture by manipulating the composition of media and growth regulators (physiological and biochemical conditions), but on the whole it is a good source for the establishment of suspension culture

Callus is formed through three stages of development, such as:

• Induction 

• Cell division and 

• Cell differentiation

Induction

During this stage, metabolic activities of the cell will increase; with the result, the cell accumulates organic contents and finally divides into a number of cells. The length of this phase depends upon the functional potential of the explant and the environmental conditions of the cell division stage

Cell division

This is the phase of active cell division as the explant cells revert to meristematic state

Cell differentiation

This is the phase of cellular differentiation, i.e. morphological and physiological differentiation occur leading to the formation of secondary metabolites 

Maintenance

After sufficient time of callus growth on the same medium following change will occur, i.e

• Depletion of nutrients in the medium 

• Gradual loss of water 

• Accumulation of metabolic toxins

Hence for the maintenance of growth in callus culture it becomes necessary to sub-culture the callus into a fresh medium. Healthy callus tissue of sufficient size (5–10 mm in diameter) and weight 20–100 mg) is transferred under aseptic conditions to fresh medium; sub-culturing should be repeated after even four to five weeks

Many callus cultures however remain healthy and continue to grow at slow rate for much longer period without sub-culturing, if the incubation is to be carried out at low temperature, 5–10°C below the normal temperature (16–18°C). Normally, total depletion takes about 28 days

Callus tissue may appear in the following different colours:

White: If grown in dark due to the absence of chlorophyll 

• Green: If grown in light 

• Yellow: Due to development of carotenoid pigments in greater amounts 

• Purple: Due to the accumulation of anthocyanins in vacuole 

• Brown: Due to excretion of phenolic substance and formation of quinones

Callus culture may vary widely in texture appearance and rate of growth. Some callus growth is heavily lignified and hard in texture while others are fragile. The cells in callus tissue vary in shape from spherical to elongated.

Suspension Culture

Suspension culture contains a uniform suspension of separate cells in liquid medium. For the preparation of suspension culture, callus fragments is transferred to liquid medium (without agar), which is agitated continuously to keep the cells separate. Agitation can be achieved by rotary shaker system attached within the BOD incubator at a rate of 50–150 rpm. After sufficient numbers of cells are produced, subculturing can be done in fresh liquid medium. Single cells can also be obtained from fresh plant organ (leaf)

Initiation of suspension culture

Isolation of single cell from callus culture: Healthy callus tissue is selected and placed in a petridish on a sterile filter paper and cut into small pieces with the help of sterile scalpel. Selected small piece of callus fragment about 300–500 mg and transferred into flask containing about 60 ml of liquid nutrient media (i.e. defined nutrient medium without gelling agent), the flasks is agitated at 50–150 rpm to make the separation of the cells in the medium. Decant the medium and resuspend residue by gently rotating the flask, and finally transfer 1/4th of the entire residue to fresh medium, followed by sieving the medium to obtain the degree of uniformity of cells.

Isolation of single cell from plant organ: From the plant organ (leaf tissue) single cell can be isolated by any of the following methods:

• Mechanical method 

• Enzymatic method

Mechanical method: The surface sterilized fresh leaves are grinded in (1:4) grinding medium (20 μmol sucrose; 10 μmol MgCl2 , 20 μmol tris-HCl buffer, pH 7.8) in glass pestle mortar. The homogenate is passed through muslins (two layers) cloth, washed with sterile distilled water, centrifuged with culture medium, sieved and placed on culture dish for inoculation.

Enzymatic method: Leaves are taken from 60- to 80-day-old plant and sterilized by immersing them in 70% ethanol solution followed by hypochlorite solution treatment, wawith sterile double distilled water, placed on sterile tile and peeled off the lower surface with sterile forceps. Cut the peeled surface area of the leaves into small pieces (4 cm2 ). Transfer them (2 g leaves) into an Erlenmeyer flask (100 ml) containing about 20 ml of filtered sterilized enzyme solution (macerozyme 0.5% solution, 0.8% mannitol and 1% potassium dextran sulphate). Incubate the flask at 25°C for 2 h. During incubation, change the enzyme solution with the fresh one at every 30 min, wash the cell twice with culture medium and place them in culture dish.

Growth pattern of suspension culture

Cell suspension culture is generally initiated by transferring an established (undifferentiated) callus tissue to a liquid nutrient medium, in flask culture vessel, which is agitated continuously during culture period. Agitation serves both, to aerate the cultures and to disperse the eel in medium. The composition of the medium for the establishment of suspension culture could be the same as for the callus culture except for the addition of agar. After transferring the cells into a suitable liquid medium they divide after lag phase and linearly increase their population. The soft callus generally forms a suspension culture without much difficulty. The release of cells and tissue fragments from less friable callus masses and the maintenance of good degree of cell separation may often be promoted by the presence of liquid medium of a high auxin concentration—an appropriate balance between yeast extract and auxin or between auxin and kinetin. After sometime depending upon the nutrient level and the rate of cell division, it comes to stationary phase

Stationary phase: The suspension culture is usually incubated at 25°C in darkness or low intensity fluorescent light at this stage, cell cultures are sub-cultured by dilution of stock culture 5–10 times (v/v) depending upon the growth of cells. The growth of suspension culture is higher than callus culture, and therefore it requires rapid sub-culture (7–21 days) as compared to callus culture (four to eight weeks). 

The incubation period from culture initiation to the stationary phase is determined primarily by:

• Initial cell density 

• Duration of lag phase and 

• Growth rate of cell type

The cell density used to sub-culture is critical and depend largely on the type of suspension culture to be maintained. The low initial cell density will prolong the lag phase and exponential phase of growth. At an initial cell density of 9–15 × 103 ml, the cell will generally undergo eightfold increases in cell number before entering the stationary phase. Normal incubation time of stock culture is 21–28 days, while for sub-culture it is 14–21 days

There are several parameters for measuring growth of cultured cells such as measurement of fresh and dry weights, cell mass, cell number, mitotic index or indirectly by the conductivity of the medium (King et al., 1973)

• Fresh weight: The value of callus cultures, frequently determined as total weight of callus medium layer and petridish. However, in this method, there are variations due to evaporation via the medium’s surface. Hence, more exact values are obtained by determining the weight after complete separation from the culture medium. This is possible when the material is cultured on separate layers of cellulose or nylon. 

•  Dry weight: It requires repeated drying usually at 60°C to the point of constant weight, up to fresh weight of 500 mg, a linear relationship between fresh and dry weight is assumed. This method excludes error due to varying endogenous water contents. 

•  Cell mass: It may be determined by densification by centrifugation (Ca 2000 g, 5 min) of a particular percentage of the volume (4–7 ml) in graduated conical centrifuge tubes. In order to avoid error, due to water absorption by the cells, the so-called packed cell volume (PCV) must be recorded immediately following the separation process, 

•  Cell number: To determine the number of cell per unit volume, existing cell clumps or aggregates must be separated into isolated cell (callus culture and in most suspension cultures). This is commonly done using chrome-trioxide alone or in combination with hypochlorous acid. Possible alternative are EDTA and pectinase. 

•  Conductivity: The inverse relationship between the conductivity and fresh or dry weight of the medium allows the determination of growth without taking samples (which would affect the sterility of the culture); in fully synthetic media, conductivity is determined almost exclusively by salt concentration. As long as the pH of the medium remains above 3, the concentration of hydrogen ions does not affect conductivity. 

•  Cellulose concentration: Calcofluor-white ST (0.1% aqueous) allows monitoring of changes in the concentration of cell wall polymers from β-glucoside bglucose molecule such as cellulose or callose. The textile brightener specifically bonds to β-1,4 glucans and intensely fluoresces following stimulation with short wave blue light. In this way even traces of these compounds may be identified.

APPLICATIONS OF PLANT TISSUE CULTURE

Plant tissue culture technology has been used in almost all the field of biosciences. The desirable products produced by plant tissue cultures are as diversified as is industry itself. Its applications include:     

• Production of Phytopharmaceuticals 

• Biochemical Conversions 

• Clonal Propagation (Micro-propagation) 

• Production of Immobilized Plant Cells

Production of Phytopharmaceuticals

The use of plant tissue culture for the production of phytopharmaceuticals was started in 1959 when Wenstein et al., studied Agave, for the production of steroids using tissue culture method. Dioscorea was reported to contain industrially useful steroids by 1966, but it was 1969, when Kaul reported the production of 1.2% dry weight diosgenin by tissue culture of D. sylvatica     

During the last two decades advancement in tissue culture technology such as development of hairy root cultures, immobilized plant cell systems, and technique to enhance the excretion of desired product into medium has resulted in promising findings for a variety of medicinally important substance from several medicinal plants. Even the callus and suspension cultures are also capable of synthesizing secondary metabolites, and yields are comparable to the intact plant as

Biochemical Conversion (Bio-transformation)

The conversion of small part of a chemical molecule by means of biological systems is termed bio-transformation. It is a process in which the substrate can be modified. For example, Digitalis lanata cell cultures have ability to effect hydroxylation, acetylation, glycosylation, etc. It is reported that D. lanata strain 291 can convert β-methyl digitoxin into β-methyl digoxin. Cell suspension culture of Strophanthus gratus affects various biochemical conversions of digitoxigenin. Monoterpene bio-conversions are reported with mentha cell culture. It can convert menthone to (+) neomenthol and pulegone to isomenthone.

Clonal Propagation (Micro-Propagation

Clonal propagation (micro-propagation) is the technique to produce entire plant from single individual by asexual reproduction. This fact can be commercially utilized to produce high-yielding crops of the desirable characters in a short period of time, which otherwise show variation when grown using seeds. For example, Foeniculum vulgare (fennel) shows wide variations in the yield and composition of the volatile oil, and by this technique, it has been reported to have uniform clones of fennel with narrow variation in the volatile oil composition, in comparison to the normal cultivation.

Somaclonal Variation

In clonal propagation, clones are produced from tissue culture with uniform characters but few clones may show variations among the population of clones, which were not present in the parent cells. This formation of variant clones from cultured tissue is called as somaclonal variations. Variants are of two types: 

• desirable variants and

• undesirable variants.

Desirable variants can be used for the improvement of crops. The clone showing high productivity can be used for commercial purposes.

Immobilization of Plant Cells

The immobilization of plant cell or enzymes has increased the utility of plant cell biotechnology for production of pharmaceuticals. The plant cells can be immobilized by using matrices, such as alginates, polyacrylamides, agarose and polyurethane fibres. The immobilized plant cells can be utilized in the same way as immobilized enzymes to effect different reactions.

Immobilized cell systems may be used for bio-conversions, such as codeinone to  codeinine and digitoxin to digoxin or for synthesis from added precursors, e.g. production of ajmalicine from tryptamine and secologanin. The suspension cultures of Anisodus tanguticus have been reported to convert hyoscyamine to anisodamine in good quantity. Subsequently, the cultures convert anisodamine into scopolamine. The bio-transformation reactions, such as glycosylations, hydroxylation, acetylation, demethylation, etc., have been successfully attempted in immobilized cell systems. The hydroxylation or glycosylation of cardiac glycosides in cultures of Digitalis lanata and Daucus carota have also been reported.

Immobilized plant cells can be used for tracing the biosynthetic pathways of secondary metabolites and also can be used for carrying out bio-transformation or biochemical reactions