Landmarks/Milestones
1 1902—Haberlandt presented the classic paper describing his pioneering attempt to culture isolated plant cells in a simple nutrient solution at a meeting of the Vienna
Academy of Sciences in Germany.
2 1904—Hannig initiated the work on excised embryo culture of several Crucifers.
3 1922—Knudson demonstrated asymbiotic in vitro germination of orchid seeds.
4 1925, 1929—Laibach demonstrated the practical application of embryo culture to produce interspecific hybrids between sex- ually incompatible parents (Linum perenne x L austriacum).
5 1934—White established continuously growing cultures of tomato root tips.
6 1937—White formulated the first synthetic plant tissue culture medium (WM).
7 1939—Gautheret, Nobécourt and White, independently, established continuously growing tissue cultures.
8 1941—Van Overbeek introduced coconut water as a medium constituent by demon- strating its beneficial effect on in vitro development of immature embryos and callus formation inDatura.
9 1946—Ball succeeded in raising whole plants from excised shoot tips of Lupinus andTropaeolum.
10 1947—Braun proposed the concept of tumor inducing principal (TiP) of Agro- bacterium tumefaciens responsible for autonomous growth of crown gall tissue.
11 1950—Braun demonstrated that Ti principal inAgrobacterium tumefaciensis transferred to plant genome naturally.
12 1952—Morel & Martin developed the tech- nique of meristem culture ofDahliato raise virus-free plants from infected individuals.
13 1954—Muir et al succeeded in inducing divisions in mechanically isolated single cells cultured in the presence of a nurse tissue.
14 1955—Miller et al discovered the first cytokinin (kinetin) from autoclaved herring sperm DNA.
15 1957—Skoog and Miller put forth the con- cept of chemical control of organogenesis (root and shoot differentiation) by manipu- lating the relative concentrations of auxin and kinetin.
16 1958—Steward (USA) and Reinert (Germany), independently, reported the formation of embryos by the somatic cells of carrot (somatic embryogenesis).
17 1960—Jones et al successfully cultured isolated single cells using conditioned medium in microchamber.
18 1960—Bergmann developed the cell plating technique for the culture of isolated single cells.
19 1960—Morel described a method for rapid in vitro clonal propagation of orchids
20 1960—Cocking isolated plant protoplasts enzymatically.
21 1962—Kanta et al developed the technique of in vitro pollination; viable seed formation by in vitro pollination of naked ovules.
22 1962—Murashige & Skoog formulated the most widely used plant tissue culture med- ium (MS).
23 1964—Guha and Maheshwari produced the first androgenic haploid plants ofDaturaby anther culture.
24 1965—Johri and Bhojwani demonstrated the totipotency of triploid endosperm cells.
25 1965—Vasil and Hildebrand achieved regeneration of full plants starting from isolated single cells of tobacco.
26 1966—Kohlenbach succeeded in inducing divisions in isolated mature mesophyll cells ofMacleaya cordatawhich later differenti- ated somatic embryos.
27 1970—Power et al published the first report of chemical fusion of plant protoplast.
28 1970—Establishment of International Asso- ciation of Plant Tissue Culture (IAPTC).
29 1971—Heinz and Mee reported somaclonal variation in the regenerants from callus cultures of sugarcane.
30 1971—Takebe et al achieved plant regen- eration from isolated protoplasts of tobacco.
32 1972—Carlson et al produced the first somatic hybrids by the fusion of isolated protoplasts of Nicotiana glauca and N. langsdorffii.
33 1973—Nitsch and Norreel succeeded in producing haploid plants from isolated microspore cultures of tobacco.
34 1973—Nag and Street succeeded in regen- eration of plants from carrot cells frozen in liquid nitrogen (-196°C).
35 1974—Zaenen et al identifiedTiplasmid as the causative factor of Agrobacterium tum- efaciensfor crown gall formation.
36 1974—Kao et al and Walin et al intro- duced PEG as a versatile chemical for the fusion of plant protoplasts.
37 1974—Reinhard reported biotransformation by plant tissue cultures.
38 1976—Seibert reported regeneration of shoots from cryopreserved shoot.
39 1976—San Noeum reported the develop- ment of gynogenic haploids from the cul- tured unfertilized ovaries of barley.
40 1977—Chilton et al demonstrated that only a part of theTiplasmid ofA tumefaciensis responsible for crown gall formation.
41 1984—Horsch et al produced the first transgenic plants of tobacco by co-culture of leaf discs withAgrobacterium tumefaciens.
42 1986—Abel et al produced the first trans- genic plants with useful agronomic traits.
43 1987—Sanford et al invented the biolistic method of direct gene transfer into plant cells.
44 1987—Fujita and Tabata developed com- mercial process for the production of shikonin by cell cultures of Lithospermum erythrorhizon.
45 1993—Kranz et al reported regeneration of full plants from in vitro fertilized eggs of maize (In Vitro Fertilization).
46 1994—Holm et al succeeded in raising full plants from excised in situ fertilized eggs (zygotes) of barley.
47 1995-To date; the existing in vitro techniques were refined to enhance their efficiency and were applied to increasing number of plant species with different objectives.
48 1995—IAPTC Newsletter developed into Journal of Plant Tissue Culture and Biotechnology.
49 1998—IAPTC renamed as International Association of Plant Tissue Culture and Biotechnology (IAPTC & B).
Association of Plant Biotechnology (IAPB).
Bhojwani SS, Razdan MK (1996) Plant tissue culture: theory and practice, a revised edition Elsevier,
Gautheret RJ (1985) History of plant tissue and cell culture: a personal account In: Vasil IK (ed) Cell culture and somatic cell genetics of plants, vol 2. Academic Press, NewYork
Krikorian AD, Berquam DL (1969) Plant cell and tissue cultures: the role of Haberlandt Bot Rev 35:59–88 Thorpe TA (2007) History of plant tissue culture Mol Biotechnol 37:169–180
Vasil IK (2008) A history of plant biotechnology: from the cell theory of Schleiden to biotech crops Plant Cell Rep 27:1423–1440
White PR (1943) A handbook of plant tissue culture. Jacques Cattell Press, Lancaster
Introduction
A plant tissue culture laboratory, essential for both research and commercial purposes, must include key facilities such as washing and storage areas for labware, preparation and sterilization zones for nutrient media, and aseptic manipulation areas for plant materials Additionally, it should maintain cultures under controlled conditions of temperature, light, and humidity, allow for observation of cultures, and facilitate the hardening of in vitro developed plants The level of sophistication in equipment and facilities varies based on specific needs and available funding, highlighting the importance of ingenuity and careful planning in establishing a new tissue culture facility.
Requirements
Structure and Utilities
Building a laboratory from the ground up is expensive, but there is flexibility in design and material selection during the conceptual phase Ideally, a commercial laboratory should be situated in an existing building with necessary modifications After assessing economic feasibility, an independent facility may be constructed Typically, tissue culture laboratories are developed from existing infrastructure, sharing equipment and facilities with other labs A well-equipped research facility should include four essential rooms: a Washing Room for glassware cleaning and storage, a Media Room for media preparation, a Sterile Area for aseptic tasks, and a Growth/Culture Room to maintain cultures under optimal conditions The Growth Room should feature a working table, a stereoscopic microscope, and efficient lighting, such as cool light from fiber optics, for culture observation Sterile transfer cabinets can be located in the culture room or a designated transfer area, often situated in a quiet section of the laboratory.
When constructing a facility for commercial use, it's advisable to position it away from urban areas to minimize exposure to pollution and vibrations This may necessitate transporting personnel to the site The laboratory should be situated away from agricultural fields to prevent contamination from machinery during harvest Additionally, the facility must be safeguarded against rain and wind, which can carry harmful spores and pests Energy conservation through thermal insulation is crucial, and one effective strategy is to place the transfer area and growth rooms below ground level, ensuring protection against water seepage and providing sufficient ventilation Alternatively, these rooms can be designed with double walls or constructed from hollow materials.
S S Bhojwani and P K Dantu, Plant Tissue Culture: An Introductory Text,
11 bricks with air trapped in between, which could be vented during summers.
A tissue culture facility necessitates a substantial supply of high-quality water, making it crucial to carefully consider the water source and wastewater disposal during the design phase This is especially important in areas lacking sewer facilities, as compliance with local municipal health and environmental regulations must be prioritized.
A tissue culture unit requires a reliable power backup system to prevent contamination of cultures during power failures or load-shedding, particularly during aseptic manipulations Without this backup, valuable cultures risk being lost due to temperature fluctuations in the growth room during electricity outages Installing a generator with a self-starting switch can ensure continuous operation and protect vital cultures from damage.
Maintaining cleanliness in a tissue culture laboratory is crucial, and the layout should minimize backtracking of materials The design must effectively separate the 'clean area', which includes the transfer and growth rooms, from the 'unclean area', treating it as a restricted zone inaccessible to visitors In commercial settings, individuals must wash their hands and feet and don sterilized overcoats and headgear before entering this restricted area to ensure optimal hygiene standards.
Maintaining high sanitation standards is crucial, especially in environments with abundant dust, pollen, and small insects Implementing paved pathways and surrounding areas with shrubs can enhance cleanliness Achieving a high level of cleanliness and minimizing contaminants can be facilitated by maintaining positive air pressure in designated 'clean areas.'
Depending on the necessity, a Class 1,000 or
Class 10,000 standard should be maintained for the clean room For the movement of material in
To maintain high asepsis in the clean area, it is essential to use sterilized mediums, instruments, and water, while keeping glassware and old infected cultures out of this space Additionally, a window with a double door hatch should be installed to enhance cleanliness and prevent contamination in the clean area, especially when handling tissue culture produced plants for hardening.
Utilizing locally sourced construction materials, equipment, and tools is essential for cost efficiency and simplified maintenance Emphasizing innovation and the use of indigenous fabrication methods can significantly lower expenses in construction projects.
Washing Room
The washing and sterilization areas in a laboratory can be either separate or combined, depending on available funds and space It's essential to have a reliable supply of hot and cold running water and a large, acid and alkali-resistant sink in the washing area Adequate steel or plastic buckets and tubs are necessary for soaking culture vials and labware, while various brushes are crucial for cleaning glassware Although a dishwashing machine is optional, an industrial dishwasher is recommended for commercial setups The media room should include a hot air cabinet for drying labware, an oven for dry sterilization, and a dustproof cupboard for storing plastic and glassware To prevent interference between washing and sterilization activities, a temporary partition can be erected, or the timing of these tasks can be staggered If an autoclave is located in the media room, it should be placed in a ventilated area.
Good quality water, despite its availability, is unsuitable for final washing of labware or medium preparation due to impurities like inorganic and organic compounds, dissolved gases, particulate debris, and microorganisms To achieve the necessary purity, water can be purified through methods such as distillation, deionization, or reverse osmosis, often requiring a combination of these techniques Water purity is typically assessed by measuring its resistivity in ohms cm -1 or its reciprocal.
12 2 General Requirements conductivity (mhos cm -1 ) Water for tissue culture should ideally have a conductivity of 5.0 lmhos cm -1 although a conductivity level up to
For research and commercial applications, it is essential to use high-quality water purification systems, such as water distillation apparatus, reverse osmosis units, or Mili-Q purification systems, rather than relying solely on deionized water, which is suitable for teaching laboratories or rinsing labware The selection among these options depends on the desired water quality, production speed, and cost considerations A glass distillation unit with a suitable handling capacity is recommended for research laboratories to ensure optimal water quality.
1.5 to 2 L h -1 of water should be sufficient For a commercial set-up or where high purity water is required a Mili-Q purification system that can provide 90 L h -1 may be used Proper storage tanks should be provided for the purified water.
Media Room
The media room serves as the central hub of the tissue culture facility, equipped with a central working table and wall-mounted benches These surfaces are designed for durability and hygiene, featuring tops made of either granite or laminated board.
Tables and benches in the laboratory should be designed for standing work, featuring storage solutions like drawers and cupboards beneath Essential equipment such as balances, pH meters, magnetic stirrers, and hot plates should be placed on these benches It is crucial to include a top-loading electronic balance for larger quantities and an analytical balance for precise measurements of small chemical amounts Additionally, if the media room contains an autoclave, the balances should be enclosed in a separate chamber For larger commercial labs, an automatic media dispenser can enhance efficiency.
For the short-term storage of specific chemicals, plant materials, and stock solutions, it is essential to have a refrigerator and a deep freeze, which can be placed in the corridor if space is limited Additionally, a single electrode pH meter capable of measuring conductivity should be available To filter sterilize media or solutions containing thermolabile compounds, an aspirator or vacuum pump may be necessary For steam sterilization, an autoclave or a domestic pressure cooker, depending on the volume to be sterilized, is required.
For emergencies a fire extinguisher and a first aid kit should be kept in this room.
Glassware/Plasticware
In a tissue culture laboratory culture vessels
For effective tissue culture work, it is essential to maintain adequate supplies of specific containers Rimless test tubes measuring 259150 mm are commonly utilized for culture initiation and establishment, especially in commercial setups For mass multiplication, larger containers like jam bottles or wide-mouthed bottles are necessary, and Erlenmeyer flasks can also serve as suitable culture vessels It is crucial to use only borosilicate or Pyrex glassware to ensure optimal results.
Plastic culture vials, which are autoclavable and presterilized, have largely supplanted glass culture vials The market now offers a variety of presterilized, disposable culture vials made from clear plastic, specifically designed for protoplast, cell, tissue, and organ culture, available under various brand names Additionally, presterilized plastic Petri dishes are also widely accessible.
Jars, screw cap bottles, and various cell culture plates are equipped with closures, while culture tubes and flasks have traditionally utilized non-absorbent cotton plugs wrapped in a single layer of cheesecloth as their sealing method.
Autoclavable, transparent polypropylene caps with a membrane built into the top are also avail- able (KimKaps, Kimble, Division of Owens, IL).
Cotton plugs offer excellent aeration but lead to rapid dehydration of the medium Conversely, polypropylene caps slow down medium desiccation but can cause moisture and gas buildup in the container It's crucial to choose closures that allow proper aseptic aeration without hindering the growth of culture materials Notably, Parafilm/Nescofilm, often used for sealing Petri dishes, releases butylated hydroxytoluene, which is harmful to cultured plants A safer alternative is cling film, as it releases 2-ethyl-1-hexanol, which does not inhibit culture growth.
You can now purchase culture vessels made from various synthetic materials, with polypropylene vessels transmitting 65% of light and polycarbonate vessels nearly 100% For sensitive plant materials, gas-permeable fluorocarbonate vessels are also available to prevent gas buildup In addition to culture vials, a range of glass and plasticware, including beakers, measuring cylinders, and pipettes in various sizes, is essential for effective media preparation.
Transfer Room
In research laboratories, transfer hoods are typically located in growth or culture rooms, or in quieter areas of general labs However, commercial facilities require distinct transfer and growth rooms to ensure optimal conditions for culture.
The transfer room must maintain a high level of cleanliness and a worker-friendly environment, with transfer hoods utilized for aseptic manipulations Culture media are often stored in this room, although a separate storage area within the clean zone is preferable Trolleys with multiple shelves are recommended for transporting culture media and cultures, and they can also serve as side benches for holding additional materials until they are moved to the growth room Given the use of fire and heat in transfer hoods, it is essential to have a fire extinguisher readily available in this area.
Growth Room
Inoculated culture vials are placed in a controlled growth room for incubation, where maintaining cleanliness is crucial This can be ensured by implementing positive air pressure or an overhead air curtain to eliminate dust The room should feature a single door and no windows to avoid external light interference, and wall junctions should be rounded to minimize cobweb accumulation Additionally, using semi or high-gloss paint on the walls and a linoleum floor will facilitate easy cleaning and maintenance.
Tiller et al (2002) reported that the polymer
Hexyl-PVP when coated on a glass surface kil- led 99 % of harmful bacteria.
Growth rooms, designed as enclosed spaces, necessitate the use of devices to manage temperature and light effectively Air conditioning is essential for temperature control; however, opting for tower air conditioning can be costly If this method is selected, it is crucial to implement proper filtration systems to ensure that dust, spores, and other contaminants do not enter the growth environment.
In research laboratories, maintaining a clean area with stable temperature and light conditions is crucial Window-mounted or split air conditioners are typically used for cooling in summer, while hot air blowers provide heating in winter, all regulated by temperature controllers to maintain a consistent temperature of 25±2°C For specialized temperature treatments, incubators equipped with fluorescent lights can be utilized, often placed outside growth rooms to prevent tampering Cool white fluorescent lamps with electronic ballasts are preferred for growth rooms due to their uniform light intensity, with cultures usually maintained in diffuse light levels of 3000–5000 lux This low light intensity can be effectively achieved by installing three tubes one foot above 3.5 feet wide shelves.
Using a sheet of aluminum foil or a coat of aluminum paint above the tubes enhances light intensity below, while it's essential to accommodate cultures that require total darkness or high light intensities Additionally, automatic timers are recommended to effectively regulate the photoperiod for optimal growth.
To maintain optimal conditions in the growth room, it is essential to use humidifiers if the relative humidity drops below 50% to prevent rapid drying of the medium Conversely, during high humidity periods, especially in the rainy season, dehumidifiers may be necessary to avoid damp cotton plugs and the condensation of water droplets, which can lead to increased contamination risks for cultures.
In growth rooms, culture vials are organized on specially designed shelving units that can be either stationary or moveable Stationary shelves may be attached to the walls or constructed as angular racks for optimal placement Alternatively, moveable racks on roller coaster wheels enhance space efficiency Open shelves are favored for improved air circulation and can be constructed from plywood or rigid wire mesh, with each shelf equipped with its own set of fluorescent tubes for adequate lighting.
In case the cultures are to be maintained under different photoperiod and temperature regimes it is advisable to have more than one growth room.
Culture flasks, jars, and Petri dishes can be placed directly on shelves or in appropriately sized trays, whereas culture tubes require support from metallic wire racks that accommodate 20–24 tubes In commercial settings, using autoclavable plastic or epoxy-coated metallic trays for transporting large quantities of culture vials is both convenient and time-efficient It is essential to label culture vials and their trays properly to prevent mix-ups, and cart trolleys can be utilized for the transportation of culture vessels.
For optimal growth of cell suspension cultures, the culture room must be equipped with a shaking machine, which can be either horizontal or rotary Additionally, shakers featuring controls for speed, temperature, and light are also available to enhance the cultivation process.
Cold Storage
In a commercial environment, maintaining cold storage at 2–4°C for temperate plants and 15°C for tropical plants is essential These cold storage rooms facilitate the breaking of dormancy in certain plant materials, help schedule workloads by storing cultures, maintain 'mother' cultures, and preserve harvested plants (Mageau 1991).
Greenhouse
To successfully grow mother plants and acclimatize in vitro produced plants, a tissue culture laboratory must include a greenhouse constructed from glass, polythene, or polycarbonate, based on budget constraints This greenhouse should be equipped with a fan and pad system to maintain high humidity levels Additionally, having a potting room located nearby would be beneficial for the overall process.
A separate autoclave might be required in this area if one wants to sterilize the potting mixture.
In a commercial laboratory provision for cer- tain other rooms such as, a general storage, and employee’s tea room, an administrative office and shipping and receiving centre should be made.
Techniques
Glassware and Plasticware Washing
To ensure proper hygiene in the laboratory, all glassware and plasticware, except pre-sterilized items, must be thoroughly washed before first use This involves soaking the apparatus overnight in a standard laboratory detergent, followed by manual or machine scrubbing with a bottle brush After rinsing under tap water, a final rinse with distilled water is essential Contaminated glass and plastic culture vials should be autoclaved prior to washing or disposal to prevent the spread of bacterial and fungal contaminants Cleaned apparatus should be placed in wire baskets or trays for optimal drainage and then dried in a hot air cabinet at approximately 75°C before being stored in a dust-proof cupboard For transporting washed labware, appropriate trays and mobile carts should be utilized.
Sterilization
In a plant tissue culture laboratory, various elements such as labware, culture medium, plant material, and even the operator can introduce infections The nutrient-rich tissue culture medium fosters rapid growth of microorganisms, including fungi and bacteria, which can outpace and ultimately kill the plant tissues Additionally, these microbes may produce toxic wastes that inhibit the growth of cultured tissues Therefore, it is crucial to maintain a completely aseptic environment within culture vessels To prevent contamination, plant tissue culture facilities should remain separate from microbiology and pathology labs, and any contaminated vessels must be promptly removed.
The various sources of contamination and measures to guard cultures against them are discussed in the following pages.
Culture vials, whether glass or plastic, are significant sources of contamination, especially with prolonged use Glass culture vials can be dry sterilized to eliminate bacteria that may survive autoclaving, and they are typically sterilized alongside the culture medium For pre-sterilized medium, glass vials should be sterilized through autoclaving or dry heating at 160–180°C for 3 hours, ensuring the oven is equipped with a fan for optimal air circulation and is not overloaded It's crucial to allow the glassware to cool before removal to prevent bacterial contamination and reduce the risk of cracking Not all plastic labware is suitable for heat sterilization; only materials like polypropylene, polymethylpentene, polyallomer, Tefzel ETFE, and Teflon FEP can withstand repeated autoclaving at 121°C, while polycarbonate vials should have limited sterilization cycles.
20 min as it shows loss of mechanical strength on repeated autoclaving Polysty- rene, polyvinyl chlorides, styrene acrylo- nitrile, are not autoclavable at all.
The tissue culture medium serves as both a potential source of contaminants and a supportive environment for their growth, making proper sterilization essential Typically, this is achieved through autoclaving, an effective method that uses steam heating under pressure During this process, culture vials filled with the medium and sealed with bacteria-proof closures are subjected to 15 psi and 121°C for 15 to 40 minutes, starting from when the desired temperature and pressure are reached To prevent contamination, it is important to cover cotton plugs and similar items with aluminum foil before autoclaving For smaller quantities of medium, alternative sterilization methods may be considered.
Fig 2.4 A horizontal autoclave (a) and a small vertical autoclave (b)
Fig 2.5 Working diagramme of a horizontal autoclave
A pressure cooker, functioning similarly to an autoclave, can be utilized for sterilization, with exposure times depending on the liquid volume (see Table 2.1) It's important to note that extended autoclaving may negatively impact the gelling of the medium Additionally, one must avoid opening the pressure valve during the cooling phase, as a rapid pressure drop can lead to vigorous boiling and overflow, potentially wetting vial closures Before opening the autoclave, ensure the pressure gauge reads zero and the temperature is no higher than 50°C.
It has been observed that 2–5 % of media get contaminated during manual pouring after autoclaving Moreover, certainBacillusbac- teria survive even after autoclaving at
110–120°C (Leifert et al 1994) It is, there- fore, advisable to incubate the sterilized cul- ture medium at 30–35°C for 24–48 h before use to ensure that it is free of contaminants.
Autoclaves are either horizontal (Figs.2.4a,
Autoclaves come in two main orientations: vertical and horizontal, each available in various sizes While vertical autoclaves can become cumbersome to operate at higher capacities due to their depth, horizontal autoclaves offer easier handling but at a higher cost The choice of autoclave type largely depends on budget and intended use Horizontal models can feature either a single or double door, with the double-door design allowing one door to open in the media room for loading and the other to open directly into the processing area.
‘clean area’, for unloading the sterilized medium.
Autoclaving has some disadvantages such as change of medium pH and breakdown of some media constituents The following components will be partly decomposed by autoclaving (Van Bragt et al 1971):
(1) Sucrose breaks down into glucose, fructose, and some laevulose; the autoclaved medium with sucrose will contain several sugars,
(2) Gibberellic acid looses 90 % of its activity,
(3) Vitamin B 1 disintegrates into pyrimi- dine and thiazol,
(4) Zeatin, urea, vitamin C, colchicine and antibiotics are thermolabile.
(5) Plant extracts loose some of their effectiveness.
Thermolabile compounds cannot be autoclaved with the nutrient medium; instead, they must be filter-sterilized The nutrient medium, minus the thermolabile components, is autoclaved in a flask and allowed to cool in a sterilized hood The thermolabile solution is sterilized using membrane filtration and then added to the cooled autoclaved medium—cooled to 50–40°C for semisolid media or to room temperature for liquid media For effective filter-sterilization, bacteria-proof membranes with pore sizes of 0.22–0.45 µm are utilized, which are placed in appropriately sized filter holders and sterilized by autoclaving after being wrapped in aluminum foil.
Table 2.1 Minimum time required for sterilization by autoclaving
Minimum sterilization time at 121 °C (min)
Fig 2.6 A filter assembly with syringe
2.3 Techniques 19 exceeding 121°C The thermolabile liquid taken in an unsterilized gradu- ated syringe is gradually pushed through the sterilized filter assembly containing the membrane The steril- ized liquid dripping from the other end is added directly to the autoclaved medium For large volumes, filter- sterilization can be carried out using a filtering set-up attached to a vacuum pump.
To ensure aseptic manipulation, it is crucial to sterilize instruments such as forceps, scalpels, needles, and spatulas by wrapping them in aluminum foil and autoclaving Additionally, during the procedure, these instruments should be repeatedly sterilized by dipping them in 95% ethanol and flaming, allowing them to cool before use Regularly changing the alcohol is essential for maintaining sterility.
Bacillus circulansstrains persist in alcohol for more than a week (Leifert and Waites
Heat generated by Bunsen burners can create eddy currents, increasing contamination risk during sub-culture Many laboratories utilize glass bead sterilizers, like Steripot, which reach temperatures of 250°C in 5–20 minutes, effectively sterilizing instruments when embedded for 5–7 minutes Additionally, infrared sterilizers are available for instrument sterilization within the hood After sterilization, instruments should be placed on a stand inside the laminar airflow cabinet, elevated slightly above the work table.
Plant surfaces can be home to various microbial contaminants, which pose a risk of infection To prevent this, it is essential to thoroughly sterilize plant material before introducing it to the nutrient medium Additionally, any tissues exhibiting systemic fungal or bacterial infections are typically removed during tissue culture processes.
Plant tissues can be effectively surface sterilized using different sterilants, with the choice of sterilant, its concentration, and treatment duration needing to be empirically established Refer to Table 2.2 for initial guidelines on this process.
Hypochlorite solutions, including sodium and calcium hypochlorite, are effective for decontaminating plant tissues, with a recommended treatment of 0.3–0.6% sodium hypochlorite for 15–30 minutes Enhancing sterilization efficiency can be achieved by adding surfactants like Triton-X or Tween 80, or by rinsing tissues in ethyl alcohol prior to sterilization It's crucial to balance the concentration and treatment duration to minimize plant tissue damage, as surface sterilants can be toxic Gentle shaking of the vial during sterilization is advised, followed by washing the plant material 2–3 times with sterilized distilled water in a laminar airflow chamber to eliminate any residual sterilant This process is essential for initiating cultures from delicate tissues such as immature embryos, endosperm, nucellus, and shoot tips.
Table 2.2 Effectiveness of some surface sterilizing agents a
Antibiotics 4–50 mg L -1 30–60 Fairly good a After Yeoman and Macleod (1977) b 20 % (v/v) of a commercial solution
Sterilization of the plant material and surrounding tissues is essential, with the explant being carefully dissected under aseptic conditions Inoculation occurs within a laminar airflow cabinet to maintain sterility Prior to inoculation, the sterilized or subcultured plant material is positioned on a presterilized ceramic tile, steel tray, or Petri plate for precise cutting to the desired size.
Ethyl and isopropyl alcohol have also been used to surface sterilize some plant tissues
Methanol should be avoided in the preparation process After a brief rinse in ethanol, the plant material can be left to air dry in a sterile hood until the alcohol completely evaporates, or, if the material is robust enough, it can be flamed.
To control explant contamination, many workers have employed antibiotics and antifungal compounds; however, the indiscriminate use of antibiotics can be ineffective since most bacteria that infect plants are gram-negative and less responsive to standard antibiotics Research indicates that combining Binomyl with mercuric chloride effectively reduces fungal infections.
Appendix I
A list of apparatus required for plant tissue culture work
4 Graduated pipettes (1, 2, 5, 10 ml)/autopi- pettes of variable volumes
5 Pasteur pipettes and teats for them
6 Culture vials (culture tubes, screw-cap bot- tles of various sizes, petri dishes, etc.) with suitable closure
7 Plastic or steel buckets, to soak labware for washing
8 Washing machine, for washing labware
9 Hot-air cabinet, to dry washed labware
10 Oven, to dry washed labware and dry heat sterilization of glassware
11 Wire-mesh baskets, to autoclave media in small vials and for drying labware
12 Water distillation unit, demineralization unit, Milli Q unit or reverse osmosis unit, for water purification
13 Plastic carboys (10 and 20 L), to store high quality water
14 Analytical balance, to weigh small quanti- ties and a top pan balance with tare facility, to weigh comparatively larger quantities
15 Hot plate-cum-magnetic stirrer, to dissolve chemicals
16 Plastic bottles of different sizes, to store and deep freeze solutions
17 Refrigerator, to store chemicals, stock solutions of media, plant materials etc.
18 Deep freeze, to store stock solutions of media for long periods, certain enzymes, coconut milk, etc.
19 Steamer or microwave oven, to dissolve agar and melt media
20 pH meter and conductivity, to adjust pH and conductivity of media and solutions
21 Autoclave or domestic pressure cooker, for steam sterilization of media and apparatus
22 Heat-regulated hot plate or gas stove, for steam sterilization in domestic pressure cooker
23 Exhaust pumps, to facilitate filter sterilization
24 Filter membranes and their holders, to filter sterilize solutions
25 Hypodermic syringes, for filter sterilization of solution
26 Medium dispenser, to pour medium
27 Trolley with suitable trays, to transport cultures, media and apparatus
28 Laminar airflow cabinet, for aseptic manipulations
29 Spirit lamp, burner, glass bead sterilizer or infra-red sterilizer, to sterilize instruments
31 Atomizer, to spray spirit in the inoculation chamber
32 Instrument stand, to keep sterilized instru- ments during aseptic manipulations
33 Large forceps with blunt ends, for inocula- tion and subcultures
34 Forceps with fine tips, to peel leaves
36 Scalpel holder and surgical blades, for chopping of explants
37 Trays or ceramic tile, on which explants is chopped inside the hood
38 Stereoscopic microscope with cool light, for dissection of small explants
39 Digital camera with suitable attachment/s for macro and micro photography
40 Table-top centrifuge, to clean protoplast and isolated microspore preparations, etc.
41 Incubator shaker, for liquid cultures
Appendix II
A list of suppliers of equipment for setting up a tissue culture laboratory a
Glassware (culture tubes, flasks, beakers, pipettes, etc.) Borosil
Khanna Construction House, 44, Dr R.G Thandani Marg, Worli, Mumbai 400018 Plasticware (beakers, test tube racks, desiccators, conical flasks, Petri plates)
856, Marshal House, 33/1, Netaji Subhash Road Kolkata 700001
AK Scientific Industries, 5531/9, Basti Harphool Singh, Sadar Thana Road, Delhi 110006
Laxbro Laxbro Manufacturing Co., W-53, MIDC, Baosari, Pune 411026 pH meter Labindia
Labtek Engineers Pvt Ltd., Vandana House, 4th floor, L.B.S Marg, Panchpakadi, Thane 400602
Mettler Metler Instruments AG, Ch-8606, Griefense, Switzerland
Balances, analytical and top pan Anamed
P.O Box no 8336, 31, Ujagar Industrial Estate, W.T.P Road, Deonar, Mumbai 400088 Mettler
Metler Instruments AG, Ch-8606, Griefense, Switzerland
Filter sterilization membranes Millipore (India) Pvt Ltd
50A, 2nd House, Ring Road, Peewja, Bangalore Tarsons
856, Marshal House, 33/1, Netaji Subhash Road Kolkata 700001
Water distillation/purifiers units Bhanu units Infusil India
C-251, V Cross, Industrial Estate, Peenaya, Bangalore 560058
National Physical Laboratory Pusa Road, New Delhi 110022 Millipore (India) Pvt Ltd 50A, 2nd House, Ring Road, Peewja, Bangalore Ion Exchange India Ltd.
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24th K.M Mathura Road, Faridabad 121003 Atlantis India Engineering Pvt Ltd.
4E/3, Jhandewalan Extension, New Delhi 110055 Autoclaves (horizontal and vertical) Nat Steel
Metal Chem Industries, 18, Crescent Industrial Estate, Kanjumarg (E), Mumbai 400042
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Hindustan Scientific Instruments Company, Hindustan House, C-9, Vishal Enclave, New Delhi 110027
Oven, hot plates, magnetic stirrers, vortex Associated Scientific and Chemicals
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Hindustan Scientific Instruments Company Hindustan House, C-9, Vishal Enclave, New Delhi 110027
Shakers Hindustan Scientific Instruments Company
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New Brunswick Scientific Co Inc.
First Floor, 24 Community Centre, East of Kailash New Delhi 110065
Trolleys for growth room, temperature controller, electronic timers, humidifiers and dehumidifiers
1442, Wazir Nagar, Kotla Mubarakpur, New Delhi 110003
Bhojwani SS, Razdan MK (1996) Plant tissue culture: theory and practice, a revised edition Elsevier,
Biondi S, Thorpe TA (1981) Requirements for a tissue culture facility In: Thorpe TA (ed) Plant tissue culture methods and application in agriculture Aca- demic Press, New York
Cassells AC (ed) (1997) Pathogen and contamination management in micropropagation Kluwer, Dordrecht Cassells AC (2001) Contamination and its impact on tissue culture Acta Hortic 560:353–359
Mageau OC (1991) Laboratory design In: Debergh PC, Zimmerman RH (eds) Micropropagation: technology and application Kluwer, Dordrecht
Pierik RLM (1987) In vitro culture of higher plants. Martinus Nijhoff, Dordrecht
Towa Optics (I) Pvt Ltd., 223, Okhla Industrial Estate, Ph II, New Delhi 11002
Air conditioners, central cooling system, window/split types
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Fisher Scientific Thermo Electron US India Pvt Ltd, A-255, TTC Idustrial Area, Navi Mumbai 400710
Sigma Chemical Company P.O Box 14508, St Louis, MO 63178, USA HiMedia
23, Vadhani Industrial Establishment LBS Marg, Mumbai 400086 Sigma-Aldrich Chemicals Pvt Ltd.
12th Floor, B Block, 148 Statesman House Barakhamba Road, New Delhi 110001 Merck Specialities Pvt Ltd
Shiv Sagar Estate A, located on Dr Annie Besant Road in Worli, Mumbai 400018, serves as a hub for manufacturers and suppliers The listed dealers primarily represent Indian companies, while each country has its own dealers and representatives for major foreign brands.
Introduction
The growth and development of an explant in culture are influenced by its genetic makeup, environmental conditions, and the composition of the culture medium, with the latter being the most easily adjustable factor The success of plant tissue culture experiments heavily relies on selecting the appropriate culture medium, which is derived from the nutritional needs of soil-grown plants and nutrient solutions utilized in whole plant culture.
Some of the earliest plant tissue culture media, such as callus culture medium of Gautheret
(1939) and root culture medium of White (1943), were based on Knop’s (1865) salt solution and
Uspenski and Uspenskaia (1925) created a nutrient medium for algae, while Knop developed a formulation for higher plant growth that included calcium nitrate, potassium phosphate, and magnesium sulfate Subsequent media formulations have largely built upon the foundational work of White.
Gautheret’s media Compositions of three popu- lar plant tissue culture media are given in
Table3.1 Of these, Murashige and Skoog’s medium (MS) has been most widely used Some other media developed for specific tissues or purposes are listed in respective chapters.
Media Constituents
Inorganic Nutrients
(i) Macronutrients The macronutrients are required in millimolar (mM) quantities Cal- cium (Ca 2+ ), potassium (K + ), magnesium
Magnesium (Mg 2+), nitrogen (NO 3 -), sulfur (SO 4 2-), and phosphorus (PO 4 3-) are essential macroelements in plant tissue culture media, typically provided through compounds like calcium nitrate, potassium dihydrogen phosphate, and magnesium sulfate Potassium and calcium can alternatively be supplied as KCl or KNO 3 and CaCl 2·2H 2 O, respectively Nitrogen, crucial for plant growth both in cultures and in nature, is primarily supplied in inorganic forms such as ammonium (NH 4 +) and nitrate (NO 3 -) ions Additionally, organic nitrogen sources like urea, amino acids (such as glutamine), and casein hydrolysate may also be incorporated into the medium to enhance plant development.
(ii) Micronutrients Some micronutrients, although required in small quantities, are essential for tissue growth in cultures They act as cofactors of enzymes Micronutrients typically include boron (BO 3 3- ; from
H 3 BO 3 ), manganese (Mn 2+ ; from MnSO 4
H 2 O), iron (Fe 2+ ; from FeSO 4 7H 2 O), zinc (Zn 2+ ; from ZnSO 4 7H 2 O), copper (Cu 2+ ; from CuSO 4 5H 2 O), molybdenum (gener- ally as MoO4 -
The media recipes utilize sodium molybdate dihydrate (Na2MoO4·2H2O) and cobalt chloride hexahydrate (CoCl2·6H2O) as sources of molybdenum and cobalt, respectively Additionally, trace amounts of iodine can be included from potassium iodide (KI) To maintain iron availability across varying pH levels, chelated iron (Fe-EDTA) is preferred, or an equimolar amount of a chelating agent like sodium EDTA (Na2EDTA) is added alongside iron sulfate (FeSO4·7H2O).
Organic Nutrients
To optimize plant tissue growth in cultures, it is essential to enrich the medium with organic compounds like vitamins and amino acids, as cultured plant cells produce these in insufficient amounts Additionally, sucrose serves as a crucial carbon and energy source in plant tissue culture media.
Vitamins and amino acids play a crucial role in the healthy growth of tissue cultures, with B group vitamins, such as thiamine (vitamin B1) and nicotinic acid, being essential coenzymes that must be incorporated into the growth medium.
Table 3.2 Stock solutions for Murashige and Skoog’s basal medium (MS) a
Glycine 400 a To prepare 1 L of medium take 50 ml of Stock I, and
To prepare the solution, dissolve 5 ml each of Stocks II, III, and IV in 450 ml of distilled water by heating and stirring continuously Next, separately dissolve FeSO4·7H2O and Na2EDTA·2H2O, then mix these solutions together Adjust the pH to 5.5 and add distilled water to reach a final volume of 1 liter Sucrose should be incorporated during the medium preparation process.
3.2 Media Constituents 29 acid (also known as niacin or vitamin B 3 ), pyridoxine (vitamin B 6 ) and myo-inositol
(sometimes referred to as meso-inositol).
Myo-inositol, a sugar alcohol, is added in a relatively larger quantity (100 mg L -1 ).
Thiamine, nicotinic acid and pyridoxine are used in the hydrochloride (HCl) form.
Other vitamins included in some media for- mulations are folic acid (vitamin M), ascorbic acid (vitamin C), riboflavin (lactoflavin, vitamin B 2 ), pantothenic acid (D-calcium pantothenate, vitamin B 5 ), biotin (vitamin H) and tocopherol (vitamin E).
Thiamine and myo-inositol are the only essential vitamins in plant tissue culture media, recognized for their crucial roles While other vitamins may be included for historical reasons or to enhance specific in vitro responses, thiamine and myo-inositol remain fundamental to successful plant tissue culture practices.
Amino acids play a debated role in plant tissue cultures, yet they are frequently incorporated into culture media Glycine, recognized as the simplest amino acid, is a prevalent component, along with L-glutamine and others, enhancing the growth and development of plant tissues in vitro.
Table 3.3 Role of culture media constituents
Potassium K + Necessary for normal cell division, and synthesis of proteins and chlorophyll Magnesium Mg 2+ Component of chlorophyll molecule
Calcium Ca 2+ Constituent of cell wall; involved in the regulation of hormone responses and could have a pre-emptive role in morphogenesis; deficiency may cause shoot tip necrosis
Amino acids, vitamins, nucleic acids, and proteins are essential components that play a crucial role in growth processes The presence of NH4+ is vital for somatic embryogenesis in cell and callus cultures, as it indirectly influences growth by affecting the pH of the medium.
Phosphorus PO 4 3- Vital for cell division; storage and transfer of energy (part of AMP, ADP and
ATP is essential for energy transfer in cells Sulphur (SO₄²⁻) is found in amino acids like cysteine, cystine, and methionine, playing a crucial role in protein structure Iron (Fe²⁺) is integral to various enzymes and acts as a respiratory electron carrier in compounds such as cytochrome and oxidative enzymes, including peroxidases and catalase Copper (Cu²⁺) is involved in oxidative enzymes like cytochrome oxidases, tyrosinase, and ascorbic oxidase, which are responsible for oxidizing phenolic substances Zinc (Zn²⁺) is a key component in the enzyme that synthesizes the IAA precursor tryptophan, and its deficiency can lead to symptoms such as rosetting and leaf chlorosis.
Molybdenum MoO 4 - Component of some plant enzymes, such as nitrate reductase, and therefore, essential for nitrogen metabolism
Boron BO 3 2- Exact role not known but implicated in enhancing the rate of sugar movement in plants
Thiamine Vitamin B 1 Involved in biosynthesis of certain amino acids; an essential cofactor in carbohydrate metabolism could have synergistic interaction with cytokinins. Ascorbic acid
Vitamin C An antioxidant, prevents blackening during explant isolation
Myo-inositol phosphatidyl-inositol plays a vital role in signal transduction, acting as a second messenger for auxins, and may function as a carrier and storage form of IAA in the form of IAA-myo-inositol ester It is an essential precursor for the synthesis of pectin and hemicelluloses necessary for cell wall formation, and it may also facilitate the uptake and utilization of ions Additionally, sucrose serves as a crucial source of carbon and energy, as well as acting as an osmotic agent.
In culture media, asparagine, serine, and proline act as organic sources of reduced nitrogen While inorganic nitrogen sources like NH4+ and NO3- typically provide adequate protection against nitrogen deficiency, the addition of amino acids may not be necessary for optimal growth.
Most plant tissue cultures cannot photosynthesize effectively due to a lack of chlorophyll, underdeveloped chloroplasts, limited CO2 availability, and insufficient light intensity Consequently, it is essential to incorporate a usable carbon source into the culture medium to support various metabolic activities.
The most commonly used carbon source is sucrose at a concentration of 2–5 % (w/v).
Autoclaving sucrose with the medium enhances tissue growth compared to filter-sterilized sucrose, as the autoclaving process hydrolyzes sucrose into more readily available sugars like glucose and fructose Sucrose serves as a vital energy source and is the primary osmotic component of the medium, with nutrient salts contributing about 20–50% to the osmotic potential, while sucrose accounts for the remainder.
Plant tissues utilize various forms of carbon, including maltose, galactose, mannose, and lactose Among these, maltose has been identified as particularly effective in promoting somatic embryogenesis in crops such as soybean, alfalfa, and rubber Additionally, maltose stands out as the optimal carbon source for androgenesis in wheat and rice.
Plant Growth Regulators
Addition of growth regulators (Table3.4) to an otherwise complete medium, containing inor- ganic and organic nutrients and sucrose called
Basal medium (BM) is essential for promoting growth and differentiation in plant tissues While explants may contain some endogenous growth hormones, exogenous supplementation is often required to achieve specific responses These growth regulators are typically needed in very small amounts, ranging from 0.001 to 10 µM The type and concentration of these regulators depend on the plant variety, the tissue type, and the specific culture stage, such as callus initiation, somatic embryogenesis induction, shoot differentiation, multiplication, or rooting.
To establish an effective tissue culture protocol for a new plant species, it is essential to test different types and concentrations of growth regulators through various permutations and combinations While growth regulator concentrations are commonly reported in mg L -1, expressing them in molar concentrations is preferable for meaningful comparative studies This approach accurately reflects the actual number of growth regulator molecules present per unit volume of the culture medium, enhancing the reliability of the results.
Auxins play a crucial role in various developmental processes in plants, such as stem elongation, tropisms, apical dominance, abscission, and rooting In tissue cultures, they are essential for inducing cell division, cytodifferentiation, and both organogenic and embryogenic differentiation Low concentrations of auxins promote root initiation, while higher concentrations lead to callus formation Commonly used auxins in tissue cultures include indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), α-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and para-chlorophenoxyacetic acid (p-CPA) IAA and IBA are naturally occurring, whereas NAA, 2,4-D, and p-CPA are synthetic auxins, along with others like naphthoxyacetic acid (NOA) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) that are also utilized in tissue culture applications.
5,6-tricholoropyridinecarboxylic acid (piclo- ram) and 3,6-dichloro-o-anisic acid (dica- mba).
IBA, NAA, and IAA are commonly utilized for rooting and, when combined with a cytokinin, promote shoot proliferation These hormones also play a role in tracheidal differentiation within cell and callus cultures Additionally, 2,4-D and 2,4,5-T are highly effective in inducing and supporting callus growth, with 2,4-D being crucial for somatic embryogenesis Dicamba is primarily used for monocots, while picloram has shown effectiveness in legumes.
Auxins are usually dissolved in a small amount of ethanol or dilute NaOH.
Cytokinins are a significant group of plant hormones, primarily consisting of N6-substituted adenine derivatives found in plants as nucleosides and nucleotides, with roots being a key site for their synthesis In nature, they play vital roles in cell division, apical dominance modification, and shoot differentiation In tissue culture, cytokinins are incorporated to stimulate cell division, induce adventitious shoot differentiation from callus, and promote shoot proliferation by alleviating apical dominance Common cytokinins used in tissue cultures include kinetin, BAP (benzylaminopurine), 2iP, and zeatin, while thidiazuron (TDZ) is also utilized but at low concentrations to avoid excessive callusing Additionally, adenine may be added as a weak cytokinin to enhance shoot formation BAP is the most efficient and widely used cytokinin in plant tissue culture, typically applied at concentrations ranging from 1 to 10 µM.
Topolins, are a new class of highly active naturally occurring aromatic cytokinins. These cytokinins, particularly meta-topolin
Table 3.4 Some plant growth regulators used in plant tissue culture media, their molecular weights (M.W.) and solvents
Indole-3-acetic acid IAA 175.2 Ethanol/1 N NaOH
Indole-3-butyric acid IBA 203.2 Ethanol/1 N NaOH
2,4-Dichlorophenoxyacetic acid 2,4-D 221.04 Ethanol/1 N NaOH a-Naphthalene acetic acid NAA 186.2 Ethanol/1 N NaOH
2,4,5-Trichlorophenoxyacetic acid 2,4,5-T 255.5 Ethanol para-Chlorophenoxyacetic acid p-CPA 100.59 Ethanol
4-Amino-3,5,6-tricholoro pyridinecarboxylic acid Picloram 241.5 Acetone
3,6-Dichloro-o-anisic acid Dicamba 221 Ethanol/Acetone
6-(Furfurylamino)-purine (kinetin) KIN 215.2 dil HCl/1 N NaOH
6-(4-Hydroxy-3-methylbut-2-enylamino)-purine (Zeatin) ZEA 219.2 dil HCl/1 NaOH 1-Phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (thiadiazuron) TDZ 220.3 DMSO
Tri-iodo benzoic acid TIBA 499.81 1 N NaOH
32 3 Culture Media, also known as mT [6-(3-hydroxybenzylamino)-purine], are gaining popularity among plant tissue culturists due to their beneficial impact on various tissue culture parameters These media enhance the rate of shoot multiplication, minimize physiological abnormalities, and improve rooting and acclimatization processes.
Cytokinins are generally dissolved in small amounts of dilute HCl or NaOH For thidi- azuron, DMSO (dimethylsulphoxide) is used as the solvent.
(iii) Gibberellins These are less commonly used in plant tissue culture There are over
Gibberellins, particularly GA 3, play a crucial role in plant development by stimulating internode elongation and meristem growth in certain species They are essential for the normal growth of plantlets derived from in vitro adventive embryos.
GA 3 is readily soluble in cold water (up to
1000 mg L -1 ) Being heat sensitive (90 % of the biological activity is lost after auto- claving), GA 3 is filter sterilized and added to autoclaved medium after it has cooled.
Ethylene (C2H4) is a unique gaseous plant hormone generated by aging and stressed tissues, as well as by organic components in plant tissue cultures when exposed to heat, oxidation, sunlight, or ionizing radiation This hormone plays a significant role in various morphogenic processes, including embryogenesis and organogenesis, though its effects can vary widely Ethylene can act as both a promoter and inhibitor of the same processes in different plant systems; for instance, it promotes somatic embryogenesis in maize while inhibiting it in rubber tree tissue cultures.
Ethrel, also known as ethapon (2-chloroethane phosphonic acid), is utilized in plant tissue culture studies for its ability to release ethylene upon decomposition While it typically inhibits growth and differentiation in plants, it can also promote somatic embryogenesis in certain cases.
Abscisic acid, a naturally occurring growth inhibitor, is often required for normal growth, development and maturation of somatic embryos.
Other Supplements
(i) Polyamines, derived through decarboxyl- ation of amino acids, have been used to promote organogenesis/somatic embryogen- esis (Mengoli and Bagni 1992; Rajam et al.
1998) Of the three polyamines (putrescine, spermidine and spermine), putrescine has proved most effective (Altman 1990; Litz 1993).
(ii) TIBA (2,3,5-triiodobenzoic acid) is an inhibitor of auxin polar transport In carrot cultures, it arrests development of somatic embryos at the globular stage.
(iii) Phloroglucinolhas been shown to promote rooting in rosaceous fruit trees such as apple.
(iv) Activated charcoal is usually added at0.5 % to culture media to promote rooting and/or to adsorb toxic exudates from cul- tured tissues.
Undefined Supplements
To promote the growth of specific calli and induce desired morphogenic responses in cultures, complex organic supplements like banana homogenate, corn milk, and yeast extract are often employed However, these crude natural products can lead to inconsistent results due to variations in their active constituents, which are influenced by the age of the source tissue and the plant genotype Therefore, while these supplements may be used in initial experiments, it is crucial to strive for replacement with defined components to ensure reproducibility and reliability in outcomes.
3.2 Media Constituents 33 as a single amino acid and arrive at a ‘‘synthetic medium’’ which contains only chemically defined compounds (Straus 1960).
Occasionally, actively growing plant tissues
Embryogenic calli and young ovaries serve as effective nurse tissues for the successful cultivation of single-cell systems, including both in vivo and in vitro developed zygotes, as well as isolated microspores in cereal species.
Gelling Agents
In static liquid cultures, tissue can die due to anaerobic conditions, necessitating the use of a gelling agent to solidify the medium Ideal gelling agents should be inert, withstand autoclaving sterilization, and remain liquid when heated for easy dispensing into culture vessels The semi-solid medium allows explants to be placed on its surface, ensuring adequate aeration However, gelling agents may contain varying levels of organic and inorganic contaminants, influenced by their processing and purification Common gelling agents in plant tissue cultures include agar, agarose, and gellan gum (such as phytagel and Gelrite).
Agar, derived from red algae such as Gelidium amansii, is the most widely utilized gelling agent This high molecular weight polysaccharide, composed of galactose molecules, is known for its ability to effectively bind water.
The firmness of gel is influenced by the concentration of agar and the pH level of the medium during autoclaving Agar begins to solidify at a temperature of 45°C and is typically incorporated into the medium at a concentration of 0.8% to 1% (w/v).
(ii) Agarose Agarose, comprisingb-D(1-3) and
3,6-anhydro-a-L(1-4) galactopyranose is derived from the advanced purification of agar, where agaropectins and their sulfate groups are eliminated, resulting in a higher cost compared to standard agar This compound is typically utilized at concentrations between 0.4% and 1.0%.
Agarose, which melts at 30°C, is ideal for testing thermolabile medium ingredients and for protoplast culture Gellan gum, a linear polysaccharide derived from the bacterium Pseudomonas elodea and available under trade names like gelrite and phytagel, is increasingly favored as an agar substitute Unlike agar that requires heating, gellan gum can be prepared in a cold solution; however, it should be added to the culture medium while stirring rapidly at room temperature to avoid clumping Additionally, the concentrations of divalent cations such as calcium and magnesium in the medium must be carefully controlled to ensure proper gelling of gellan gum.
Gelrite or phytagel is a good alternative to agar not only because of its low cost per litre of medium (0.1–0.2 % is sufficient) but also for many other advantages it offers.
It sets as a clear gel, which facilitates easy observation of cultures and detection of any contamination Unlike agar, their gelling strength is unaffected over a wide range of pH.
Increasing the concentration of gellan gum in the medium from 0.4% to 0.8% significantly enhances the maturation of somatic embryos in Larix eurolepsis, as noted by Teyssier et al (2011) This adjustment results in embryos exhibiting lower osmotic potential while achieving greater dry weight.
(iv) Isubgol Indian scientists have shown that the husk ofPlantago ovataseeds (Isubgol), used at 3 %, could be a cheaper alternative to gel plant tissue culture media (Jain andBabbar 1998).