BSTC-363 Cell & Tissue Culture

Biotech Online. BS (Biotechnology & Informatics).
NOTES: Cell & Tissue Culture. BSTC-363 .

References: Introduction to plant:
By H. S. Chawla.
BSTC-363 Cell & Tissue Culture.
Biotech Online. BS (Biotechnology & Informatics). Plant and Tissue Culture Micro propagation
Micro propagation: Is the production of whole plant from small sections of plant such as stem tip, node, meristem, embryo or even a seed. Plant tissue culture: is basically the same thing except that it implies the use of callus tissue generated from plant cells that are cultured in-vitro. Callus is a soft tissue composed of unorganized and undifferentiated group of cells. Micro propagation and plant tissue culture are used to produce large number of plants from small pieces of stock plant in relatively short periods of time. Why does Micro propagation work? Plant have the ability to reproduce the whole plant from single cells this is called totipotency. Totipotency is the ability of a single cell to express the full genome of in the cells to which it gives rise by cell division. Totipotency in reference to fertilized eggs like zygotes are totipotent because they produce a population of differentiated cell forming an entire organism, where as for example human skin cells are not totipotent since in culture they divide to produce only skin cells but not nerves cells or muscle cells. Plants have the ability to reproduce asexually. It is this natural ability that is the basis of micro propagation. Where does the new growth come from in plants? Meristematic tissue. Parenchyma tissue. Adventitious growth. And virtually any plant cell can be involved in the new growth of a plant. Meristematic tissue are undifferentiated cells from shoot and root tips that have not been programmed for their ultimate development Parenchyma tissue is the most common type of plant cell which can regenerate and differentiate to initiate the growth of new and varied tissues and organs. Adventitious growth is the development of new shoots, root, buds or leaves from atypical or unusual locations.
Biotech Online. BS (Biotechnology & Informatics). There are two types of plant cells division which include somatic cells and sex cells: The first one is mitosis that includes the development of somatic cells. The second one is meiosis that includes the development of sex cells. In mitosis every cell is diploid with two sets of chromosomes, the chromosomes duplicate and then segregates. The total number of chromosomes remains the same in mitosis. Here is the representation of the entire of mitosis
In prophase chromosomes replicate to form chromatids
In metaphase chromatids line up along the center.
In anaphase chromatids pull apart and move toward poles
In telophase the nucleus divide into two cells.
Meiosis or reduction division is the process of forming sexually reproductive cells. The number of chromosomes becomes half in this process. The chromosomes segregate so that one set goes to each of new sex cells or gametes which are now haploid. Here is the representation of the entire process of meiosis The process consists of meiotic division 1 and meiotic division 2.
Biotech Online. BS (Biotechnology & Informatics). Why do we perform plant tissue culture? 1. 2. 3. 4. 5. 6. 7. 8. To regenerate plants from single cell or plant tissues. To cultivate under sterile condition (in-vitro) To produce large quantities of identical plants. To cultivate without the impact of environmental conditions. To species from extinction. To isolate disease from plants. To produce plant with enhanced stress or pest resistance. To create new plant varieties (new verities can also be created by using other technologies like genetic engineering or breeding but plant tissue culture provides a plate form for the production of multiple copies of those new verities ) 9. And finally! The most important thing we can use it to make money! (But! Its more like gambling you can lose a lot of money as well!) so dont get over excited you are not going to be a millionaire by learning it. What part of plant can be used for micro propagation? Meristems, shoot and root tips, leaf tissue, anthers, embryos, flowers, virtually all parts of a plant can be used. The only limitation is that each plant is propagated differently and not every plant will respond the same way. Each genus species and variety may require a different tissue which will obtain the best results. Micro Propagation has two general forms of plantlet production: Callus production Micro cuttings Callus production is the dedifferentiation of plant cell into callus. Dedifferentiation is a cellular process in which a differentiated cell loses its special form or function, or reverts to an earlier developmental stage. Callus is then expanded into a large mass of undifferentiated cells. And then callus is activated, by selected use of plant growth hormones to redifferentiate to produce, shoot, roots and ultimately, plantlets. In Micro cuttings small sections are cut from the mother plant and placed into media, which are grown out or subdivided, again to produce more plants.
Biotech Online. BS (Biotechnology & Informatics). There are four basic stages in the process of micro propagation: Stage one is the explant establishment or initiation. Stage two is the multiplication Stage three is the rooting Stage four is acclimatization or hardening off Acclimatization: means making plant to adopt the environmental conditions.
Conclusion: Micro propagation is the process of making whole new plants by using small sections of plants Plant tissue culture implies the use of callus tissue generated from plants and are cultured in vitro The ability to produce whole new plant from single cell is known as totipotency. New growth comes from in plant by: meristematic tissue Parenchyma tissue Adventitious tissue Virtually any plant cell Plant cells can divided by means of mitosis or meiosis Micro propagation has two general forms of plantlet production Callus production Micro cutting
Biotech Online. BS (Biotechnology & Informatics). Nutrition media: The greatest progress toward the nutrition media for plant cells grown in culture took place in 1960s and 1970s.The nutrition compositions varies according to the cell, tissue, organs, and protoplast and with respect to particular plant species. Thus in a discussion of nutrition media it is important to recognize what type of culture i.e. callus, cell, organ or protoplast is to be studied and what are the objectives and purposes of this investigation. Different types of cultures have unique requirement for one or more nutritional components. a wide variety of salt mixtures have been reported in various media. A nutrient medium is defined by its mineral salt composition, carbon source, vitamins, phytohormones and other organic supplements. When reference is made to a particular medium, the intention it to identify only salt composition unless otherwise specified. Any number and concentration of amino acids, vitamins, growth regulators or organic supplements can be added in infinite variety of compositions to a given salt composition to achieve the desired results. Facilities and Equipment: Autoclave or pressure cookers. Balances, preferably electronic Centrifuge tabletop Cotton plugs, aluminum foil, brown sheets etc. Culture vessels (flasks, tubes, Petri dishes, jars, etc.) Desiccators. Dissecting microscope. Filter units. Flasks, beakers, pipettes, etc. Freezer. Hot plate/gas plate. Lab carts/trolley, trays, etc. Laminar airflow hoods. Magnetic stirrer (with hot plate). Microwave oven. pH meter. Refrigerator. Shaker. Vortex. Water bath with temperature control. Water distillation unit.
Biotech Online. BS (Biotechnology & Informatics). Units for solution preparation: The concentration of a particular substance in the media can be expressed in various units that are as follows: Unit in weight: It is represented as milligram per liter (mg/l). 10 rise to power minus six equal 1.0 milligram per liter. 10 rise to power minus seven equal 0.1 milligram per liter. 10 rise to power minus six equal 0.001 milligram per liter or I micro gram per liter. Molar Concentration: A molar (M) solution contains the same numbers of grams of substances as it is given by its molecular weight. 1 molar (M) = the molecular weight in gram per liter. 1 milli molar = the molecular weight in milligram per liter. 1 micro molar = the molecular weight in micro gram per liter.
How do you prepare 1 milliMolar Sodium Hydroxide?

First of all we have to find out the molar mass Molar mass of Sodium is 22.99 Molar mass of Oxygen is 16.00 Molar mass of Hydrogen is 1.01
So the molar mass of
Sodium Hydroxide is 22.99+16.00+1.01=40g
Molarity is moles per liter Since the molar mass of NaOH is 40g/mol, a 1 M solution of NaOH would be 40 g Add .40 grams solid NaOH to 1 liter of water and you will have 1 millimolar solution of Sodium Hydroxide Now How to prepare 2 millimolar solution of Sodium Hydroxide?
1 molar Sodium of water so 2 M
Hydroxide contains 40 grams of Sodium Hydroxide per every liter
Sodium Hydroxide contains twice that amount, i.e. 80 grams per liter.

Conversion of milligram per liter to milli moles:
For example there is 1500 milligram of potassium chloride in a solution now we know that molar mass of potassium is 39.10 and that of chlorine is 35.45, so the total molar mass
would be 74.55. Now divide, 1500, with, 74.55, and we will have the answer, 20.12. Now we are able to find out, how much millimoles of potassium and chlorine were there, in 1500 milligram of potassium chloride? To find out the amount of potassium, in the solution, multiply 20.12 with, 39.10 the answer would be, 786.692 millimoles. & to find out the amount of chlorine in the solution, multiply 20.12, with, 35.45 the answer would be, 713.254 milli moles. And if you add these two values you will have the answer 1499.964 milligrams which is approximately equal to 1500 milligrams.
Biotech Online. BS (Biotechnology & Informatics). Plant cell, tissue, and organ culture: Key factors for the manipulation of plant cell tissue and organ cultures are; 1. Nutrient media. 2. Culture explants. 3. Culture growth environments. These factors are experimentally determined to optimize growth and development including regeneration. Nutrient Media: It is composed of; 1. Inorganic salts/ mineral nutrients. 2. Organic constituents. Inorganic Nutrients: Mineral elements are very important in the life of plant for example calcium is an important component of cell wall, nitrogen is an important par of amino acids, proteins, and vitamins and magnesium is a part of chlorophyll molecules similarly iron zinc and molybdenum are of certain enzymes. Besides Carbon, Hydrogen, Oxygen and Nitrogen 12 other elements are essential for plant growth according to the recommendations of international association of plant physiology, the elements required by plants greater than 0.5mili moles per liter are referred to as macro elements and those required in concentrations less than that are referred to as micro elements. The following are required in macro milimole quantities nitrogen, potassium, phosphorus, calcium and magnesium. The essential micro elements those are required in micro molar quantities in clued iron, manganese, boron, cobalt, molybdenum, copper, zinc and iodine. For achieving maximum growth rate, the optimum concentration of each nutrient varies considerably. The active factor in the medium is the ions of different types rather than the compounds. Therefore a useful comparison between the two media can be made by looking into the total concentration of different types of ions in them. Carbon and energy source: Without exception the standard carbon source is sucrose or glucose fructose can be used but it is less efficient. The sucrose in the medium is rapidly converted into glucose and fructose. Glucose is utilized first followed by fructose. Sucrose is generally used at concentration of 2 to 5 percent. Other carbohydrates which have been tested include lactose, maltose, galactose and starch, but these compounds are generally much inferior to sucrose or glucose. Most media contain myo-inositol at a concentration of Ca 100mg/l, which improves cell growth. Vitamins: Normal plant synthesizes vitamins required for growth and development. But plant cells in culture have a requirement for vitamins. There is an absolute requirement for vitamin B1 (Thiamine). Growth is also improved by addition of nicotinic acid and
Biotech Online. BS (Biotechnology & Informatics). vitamin B6 (pyridoxine).Some media contain pathogenic acid, biotin, folic acid p- amino benzoic acid, choline chloride, riboflavin and ascorbic acid. Growth Regulators: Hormones are organic compounds naturally synthesized by higher plants which influence growth and development. Apart from natural compounds, synthetic compounds have been developed which corresponds to natural ones. These are collectively called growth regulators. There are two main classes of growth regulators that are of special importance in plant tissue culture. Auxins: A common feature of auxins is their property to induce cell division and formation of callus. Auxins cause cell division, cell elongation and swelling of tissues and the formation of adventitious roots. It often inhibits adventitious and auxillary shoot formation. At low auxins concentration adventitious root formation predominates, where as at hight concentration the root formation fails to occur and callus formation take place. The compound most frequently used and highly effective is 2,4 dicvhlorophenoxy acetic acid (2,4 D) other auxins in use include Napthelene acetic acid (N A A), indol acetic acid, indol butyric acid (I B A) etc. Cytokinins: Cytokinins are derivatives of adenine and have an important role in shoot induction. The compounds most commonly used are Kinetin benzyl adenine (B A) or Benzyle Amino Purine (B A P) etc these are often used to stimulate growth and development. They usually promote cell division if added together with auxins at higher concentrations (1 to 10 mg/l), adventitious shoot formation is induced but root formation is generally inhibited. They promote auxillart shoot formation by decreasing apical dominance. Other Hormones: Gibberellins are normally used in plant regeneration. GA3 is essential for meristem culture of some species. In general gibberellins induce elongation of inter nodes and growth of meristems and buds in vitro .Gibberellins usually inhibit adventitious root as well as shoot formation. Abscisic acid is am important growth regulator for the induction of embryogenesis. Organic Supplements: Organic nitrogen: Culture cell are normally capable of synthesizing all required amino acids but it is often beneficial to include organic nitrogen in the form of amino acids such as glutamine and aspergine and nucleotides such as adenine. For cell culture is good to add 0.2 to 1.0 g/l of casein hydrosylate or vitamin free casamino acids. The amino acids when added should be used with caution since they can be inhibitory. The amino acids included in the media and amount in milligram per liter are Glycine (2), Aspargine (100), Tryosine (100), Arginin (10) and Cysteine (10).
Biotech Online. BS (Biotechnology & Informatics). Organic acids: Plants are not able to utilize organic acids as a sole carbon source addition of acids such as citrate, malate , succinate or fumarate permits growth of plant cell on ammonia as the sole nitrogen source. The cell can tolerate a concentration of up to 10 millimoles of the acid. Pyruvate may also enhance growth of cells cultured at low density. Complex Substances: A variety of extracts protein hydrosylate yeast extracts, malt extracts, coconut milk, orange, tomato juice have been tested . Coconut milk is commonly used at 2.15 % (v/v). The present trend is however towards fully defined media and use of complex mixture is losing. Gelling agent: Agar a sea weed is the most popular solidifying agent it is a polysaccharide with high molecular mass and has the capability of gelling media. Solubilized agar forms a gel that can bind water and adsorb compounds. The higher the agar concentration, the stronger the binding of water. Plant tissue culturists often use Difco Bacto agar at a concentration of 0.6 to 1.0 % (w/v) although other forms of agar (agarose, phytagar, flow agar are also becoming popular.) pH: pH determines many important aspects of the structure and activity of biological macro molecules . Nutrient medium pH ranges from 5.0 to 6.0 for suitable in vitro growth of ex-plant. pH higher than 7.0 lower than 4.5 generally stops growth and development . pH before autoclaving is different is generally falls by 0.3 to 0.5 units after autoclaving. pH higher than 6.0 give a fairly hard medium and pH below 5.0 does not allow satisfactory gelling of the agar.
Biotech Online. BS (Biotechnology & Informatics). Sterilization Techniques Sterilization is the process used for the elimination of microorganisms. The maintenance of aseptic or sterile conditions is essential for successful tissue culture procedures. The need for asepsis requires that all the culture vessels, media and instruments used in handling tissues as well as explants itself be sterilized. In general different sterilization procedures can be groups under three categories:
1. Preparation of sterile media, container and small instruments. 2. Maintenance of aseptic conditions. 3. Preparation of sterilized ex plant material.
Preparation of sterile media, container and small instruments: Steam Sterilization: It means sterilization by using autoclave. The standard conditions for autoclaving media are 121 degree with pressure of 15 psi for 20 minutes. These conditions are followed for test tubes, or other containers containing b/w 20 to 50 ml of nutrient media. An autoclave has a temperature range of 115 to 132 degree Celsius. Good sterilization relies on time, pressure, temperature and volume of the object to be sterilized.
1. Test tubes flasks containing b/w 20 to 50ml nutrient media should be sterilized for 20 minutes at 121 degree Celsius on 15 psi. 2. Test tubes flasks containing b/w 20 to 500ml nutrient media should be sterilized for 25 minutes at 121 degree Celsius on 15 psi 3. Test tubes flasks containing b/w 50 to 5000ml nutrient media should be sterilized for 35 minutes at 121 degree Celsius on 15 psi
It must be realized that heat penetration is very important in autoclaving and larger volumes should be sterilized for longer periods as compare to smaller volumes. When autoclaving bottles their caps should be loose and after autoclaving they should be transferred to laminar air flow and their tops should be tightened once that get cool. The advantages of autoclaving are speed, simplicity, and destruction of viruses while the disadvantage is that after autoclaving the pH drops to 0.5 or 0.3 units. During and after autoclaving following should be considered:
1. 2. 3. 4. 5. pH of the media is lowered by 0.5 to 0.3 units. Autoclaving at too high temperature cam caramelize sugars, which may be toxic. Volatile substances can be destroyed by the use of autoclave. Autoclaving for too long periods can precipitate salts, and depolymarize the agar. Care should be taken to use the correct duration and temperature. Dry sterilization: The sterilization of glassware and metallic instruments can be carried out in dry heat for 3 hours at 160 degree to 180 degree Celsius. An exposure to 160 degree dry heat for 2 hour is regarded as equivalent to moist heat (steam) sterilization at 121 degree for 15 minutes. Dry goods (instruments, empty glassware) can either be wrapped in aluminum

foil brown paper or sealed metal containers to maintain sterility. Present day autoclaves come with dry and steam sterilization programmes. Filter Sterilization: Some growth factors, amino acids, vitamins and toxins are labile and get destoryed during autoclaving with the rest of natural medium. Such chemicals are therefore sterilized by filtration through a sieve or filtration assembly using filter membranes for 00.45 to 0.27 micro meters. A range of bacteria proof filters and associated equipments are available for sterilization of different volumes of liquid in the range of 1 to 200ml. Most filters are of cellulose acetate or cellulose nitrate are available in pre sterilized, plastic disposable units. During filter sterilization, all the particles, microorganisms and viruses that are bigger that the pore diameter of the filter, used are removed. Ultra violet sterilization: It is extremely expensive. The pre sterilization disposable plastic ware is generally UV radiation sterilized .the sterilized autoclave medium is then dispensed into these sterilized containers in the laminar air flow cabinet. Radiation treatments as such are not used in research laboratories. Maintenance of aseptic conditions: Alcohol Sterilization: It is essential that workers hands be relatively aseptic during manipulation work. A wash with an antibacterial detergent followed by 70% ethanol on hands is quite effective laminar air flow cabinets should be sprayed with 70 to 80% ethanol before use. Flame Sterilization: Instruments are soaked in 70 to 80% alcohol followed by flaming on burner in the laminar air flow hood, which should be carried out repeatedly while aseptic manipulation work is in progress. This is essential even if the equipments have been subjected to prior dry or steam sterilization. The precautions to be followed are: 1. Culture containers should be covered as quickly as possible after an operational step is completed. 2. Talking in hood should be avoided. 3. Set up all materials including containers, media etc in one side of the cabinet. Sterilization of Ex plant: Chemical sterilization: Plant material can be sterilized by a variety of chemicals it is the eradication of microorganisms with the aid of chemicals. The type and concentration of sterilant

to be used and exposure time, must be decided. Some of the commonly used sterilants and their effectiveness are represented in the following table: Sodium hypochlorite Calcium hypochlorite Hydrogen peroxide Silver Nitrate Antibiotics Mercuric chloride Bromide water Very effective Very effective Very effective Effective Effective Satisfactory Very effective
Protocols: Seed sterilization: 1. Wash the seeds with running tape water with few drops of detergent. 2. After that treat them with 70 % alcohol for 30 seconds to 20 minutes in a beaker. 3. The treat the seeds with 20 % to 40% sodium or calcium hypochlorite. 4. In sterile laminar hood, wash the seeds thoroughly 5 times with sterile distilled water.
Sterilization of buds, stem, leaves, roots, tubers, scales etc:

1. Treat the tissues for 30 seconds for 2 minutes in 70% alcohol by submerging in it after that wash thoroughly with running distilled water. 2. Then treat them with 20% of sodium hypochlorite solution for 5 to 10 minutes. Time varies depending on the degree of contamination. 3. In a sterile laminar air flow hood wash thoroughly 3 to 5 times with distilled water.
Sterilization of immature embryos etc:

1. They are surface sterilized first. 2. Tissue is then sub merged in 20 % sodium hypochlorite contain 2 to 3 drops for 5 to 10 minutes (stirring). 3. In the laminar air flow wash it thoroughly 3 to 4 times 4. Dissect out, excise the required ex-plant.
Cytodifferentiation: The cells in a callus are parenchymatous in nature; the differentiation of these cells into a variety of cells is required during re-differentation of cells into whole plants. This redifferentiation of cells is known as cytodifferentation. Organogenic Differentiation: For regeneration of a whole plant from a cell or callus tissue cytodifferentiation is not enough there should be differentiation leading to shoot, buds or embryo formation. This may occur through organogenesis or somatic embryogenesis. In organogenesis a monopolar structure that has a connection with the pre-existing vascular tissue within the callus, while in embryogenesis a bipolar tissue with no connection with the maternal callus tissue or ex-plant is formed. Types of Cultures: Plant tissue culture which covers all types of aseptic plant cultures should be used in a restricted sense, and it is possible to distinguish between various types of cultures. Seed Culture: Culture of seeds in vitro to generate seedling/plants. Embryo Culture: Culture of isolated mature and immature embryos. Organ Culture: Culture of isolated plant organs, different types can be distinguished, e.g. meristem,shoot tip, root culture, anther culture. Callus Culture: Culture of differentiated tissues from ex-plants allowed to de-differentiate in vitro and a so called callus tissue is produced. Cell Culture: Culture of isolated cells or very small cell aggregates remaining dispersed in the liquid media. Protoplast Culture: Culture of plant protoplast that is cells devoid of their cell walls.
Biotech Online. BS (Biotechnology & Informatics). Seed Culture: Seed culture is an important technique when ex-plants are taken from in vitro derived plants and in propagation of orchids. Sterilizing procedures are needed for plant materials that are to be used directly, as ex-plant source can cause damage to the tissues and affect re-generation. In that case, culture of seeds to raise sterile seedlings is the best method. Orchid seeds are propagated vegetatively as well as generatively. Orchid cloning in vivo is a slow process. Thus seeds can be generated in vitro and meri-cloning (that is, vegetative propagation by meristem culture) is then carried out on large scale. Most orchids are sown in vitro because;
1. Orchids seeds are very small and contain very little or no food reserves. The cells of an embryo have a simple structure and are poorly differentiated. 2. Sowing in vitro makes it possible to germinate immature orchid embryos, thus shortening the breeding cycle. 3. Germination and development takes place much quicker in vitro since there is conditioned environment and have no competition with fungi or bacteria.
Orchids seeds imbibe water via testa, and becomes swollen. After cell division has begun, the embryo cracks out of the seed coat. A proto-corm like structure is formed from the clump of cells and on this a shoot meristem can be distinguished. Protocorm has a morphological state that lies between an undifferentiated embryo and a shoot. Proto-corms obtained by seed germination have many close similarities with those produce from isolated shoot tips; the proto-corm like bodies has been introduced when cloning orchids by meristem culture. The vegetative propagation of orchids follows culture of seeds, transformation of meristem into proto-corm like bodies, the propagation of proto-corms by cutting them into pieces and the development of these proto-corms to rooted shoots. Mineral requirement of orchids The mineral requirement of orchids is generally not high and a salt poor medium of Knudson (1946) and vacin and went (1949) are good. Some of the orchids (paphiopedilum ciliolare) require darkness for germination, while others require low irradiance. Sugar is extremely important as an energy source, regulators are usually not necessary for seed germination and their addition leads to unwanted effects like callus formation etc.
Biotech Online. BS (Biotechnology & Informatics). Selection of medium: In most cases a standard basal plant medium with major salts and trace elements may be utilized. Mature embryos can be grown in a basal salt with a carbon energy source such as sucrose. But young embryos in addition require different vitamins, amino acids and growth regulators and In some cases natural endosperm extracts. Young embryos should be transferred to medium with high sucrose concentration ( 8 to 2%), which approximate the osmotic potential of the intracellular environment of the young embryo sac, and the combination of hormones which supports the growth which supports the growth of heart stage embryos (a moderate level of auxins and low level of cytokinins) Reducing organic nitrogen, asparagines, glutamine or casein hydrosylate and aelic acid are very important for growth. After one or two weeks when embryo stops to grow, it must be transferred to a second medium with normal sucrose concentration, low level of auxins and moderate level of cytokinins which allows the renewed embryo growth with direct shoot formation in many cases.
Biotech Online. BS (Biotechnology & Informatics). Embryo Culture: Embryo culture is the sterile isolation and growth of an immature or mature embryo in vitro with the goal of obtaining a viable plant. The first attempt to grow the embryo of angiosperm was made by Hannig in 1904 who obtained viable plants from in vitro isolated embryos of two crucifer cochlear and Raphanus. In 1924 Dietrich grew embryos of different plant species and established that mature embryos grew normally but those excised from immature seeds failed to achieve the organization of mature embryo. They grew directly into seedling, skipping the stages of normal embryogenesis and without the completion of dormancy period. Embryo culture is now well established branch of plant tissue culture. There are two types of embryo culture;
1. Mature Embryo Culture: It is the culture of mature embryos derived from ripe seeds. This type of culture is done when embryos do not survive in vivo, or become dormant for long periods of time, or is done to eliminate the inhibition of seed germination. Seeds dormancy of many species is due chemical inhibitor or mechanical resistance present in the structures covering the embryo, rather than dormancy of embryonic tissue. Excision of embryos from testa and culturing them in the nutrient media may bypass such seed dormancy. Some species produce sterile seeds which may be due to incomplete development of embryo. Embryo cultures may yield viable seedlings. Embryo excised from the developing seed at or near the mature stage is autotrophic and grow on simple organic medium with supplemental energy source. 2. Immature Embryo Culture:
It is the culture of immature embryos to rescue the embryos of Wide crosses. This is mainly used to avoid embryo abortion with the purpose of producing viable plant. Wide hybridization where individuals from same genus or different genera are crossed, often leads to failure. There are several barriers, which operate at pre and post fertilization levels to prevent the successful gene transfer from wild into cultivated species. The pre fertilization barriers include all factors that hinder effective fertilization, which is usually due to the inhibition of pollen tube growth by stigma or upper style. Post fertilization hinders or retards the development of zygote after fertilization and normal development of the seed. This frequently results from the failure of the hybrid endosperm to develop properly, leading to starvation and abortion of hybrid embryo, or results from embryoendosperm incompatibility where the endosperm produces toxins that kill the embryo.
Biotech Online. BS (Biotechnology & Informatics). Applications of embryo culture:

1. Preventions of embryo crosses in wide crosses. Successful inter-specific hybrids have been seen in cotton, tomato, barley and well known inter-generic hybrids include wheat X barley, wheat X ray, and barley X ray etc. embryo rescue techniques has been successfully used for raising hybrid embryo between Actidinia Delicosa X A. Eriantha. Some of the hybrids plants raised by embryo culture have recombined desired genes such as earliness, disease and pest resistance. Embryo culture is also used in crosses between diploids and tetraploids 2. Production of haploid: Embryo culture is also utilized in the production of haploids or mono-ploids. 3. Overcoming seed dormancy: Embryo culture is a technology to break seed dormancy. Seed factors including; endogenous inhibitors, specific light requirements, low temperature, dry storage requirements and embryo immaturity. 4. Shortening of breeding cycle: There are many species that exhibit seed dormancy that is often localized in the seed coat and or in the endosperm. By removing these inhibitors seeds germinate immediately seeds some time take up water and oxygen very slowly or not at all through the seed coat and so, germinate very slowly if at all. For example Brussels sprouts rose and apple etc. 5. Prevention of embryo abortion with early ripening stone fruits: Seed sterility may be due to incomplete embryo development which results in the death of germinating embryo. Embryo culture has been practiced as a general method in horticulture crops including avocado peach, nectarine and plum. Two cultivars (Gold crest peach) & (Mayfine Nectarine) have resulted from embryo culture and are now commercially grown. 6. Embryos are excellent material for in-vitro clonal propagation. This is especially true for conifers and members of graminacae family. 7. Germination of seeds of obligatory parasites without host is impossible in vitro, but is achievable with embryo culture.
Callus Culture: Callus in principle is a non-organized and little differentiated tissue. The cells in callus are of parenchymatous nature. When critically examined, callus culture is not homongenous mass of cells because it is usually made up of two types of tissue differentiated and non-differentiated. Ex-plant tissue is a differentiated tissue (roots, stem, leaves flowers etc.) which is used as a starting material for callus induction. These explants tissues generally show distinct cell division, cell proliferation and organization into specialized structures such as vascular systems. If there are only differentiated cells present in an isolated ex-plant then differentiation must take place before cell division

occurs. Parenchyma cells present in the explants usually undergo this differentiation. If the explants already contain meristmatic tissue when isolated, then this can divide without differentiation taking place. De differentiation plays a very important role, enabling mature cells in an ex-plant isolated from an adult plant to be re-determined. Callus formation: It takes place under the influence of exogenously supplied growth regulators present in the nutrient medium. The type of growth regulator and its concentration in the medium depends strongly on the genotype and endogenous hormone of the ex-plant. These requirements can be put into three categories; 1. Auxins alone (especially in monocotyledons). 2. Cytokinins alone. 3. Both auxins and cytokinins (carrot).
If the callus is difficult to induce or juvenile callus is needed, then immature embryos or seedlings or part of these are used. Factors affecting callus formation: Many factors are important for callus formation genotypes, composition of nutrient medium, physical growth factors (light & temperature) etc. MS medium or modifications of it are often used. Sucrose or glucose (2 to 4%) is usually employed as the sugar source. Light may be required in some cases and darkness in other cases. A temperature of 22 to 28 degree is advantageous for callus formation. The callus growth within a plant species may also depend on factors such as the original position of the ex-plant and the growth conditions. Sub culturing: The callus is further grown in new medium which is known as sub culturing. When sub cultured regularly on agar medium, callus cultures will exhibit an Sshaped or sigmoid pattern of growth during each passage. There are five phases of callus growth:
1. 2. 3. 4. 5. Lag phase: where cell prepare to divide. Exponential phase: where the rate of cell division is highest. Linear phase: where cell division slows and the rate of cell expansion increases. Deceleration phase: where the rates of cell division and cell elongation decreases. Stationery phase: callus growth can be monitored by fresh weight measurements, which are convenient for observing the growth of cultures over time in nondestructive manner. Dry weight measurements are more adequate than fresh weight but this method requires sacrifice of samples. A mitotic index measurement of cell division requires extensive sampling to reduce errors.
Organ culture: It is an isolated organ grown in-vitro it can be given different names (meristem, shoot tip culture, endosperm or ovule culture etc). The culture of plant organs result in three types of culture;
1. Organized : The culture of whole plant (embryos or seeds) is termed as organized culture. In this characteristic organized structure of plant or organ is maintained. If the organized culture is not broken down, then progeny arise which is identical to the original plant material (for example meristem). 2. Non-organized: If cells or tissues are isolated from an organized part of plant to de-differentiate and are then cultured, a non organized growth in the form of callus occurs. If callus disperses into the clump of cells and or single cell results, it is referred to as suspension culture. Non organized cultures have very low genetic stability. 3. Non organized/organized: Cell in an isolated organ/tissue is first de-differentiated and then re-differentiate to form organs (root or shoots, or embryos). Two organized structure can develop from nonorganized cultures either through special technique or spontaneously. In this the progeny are not often completely identical to the original plant material.
Cell culture: The growing of an individual cell which has been obtained from the ex-plant tissue or callus is referred to as suspension culture. They are initiated by transferring pieces of explant or callus into flasks with liquid medium and are then placed on a magnetic shaker to provide aeration and dispersing of cells.
Biotech Online. BS (Biotechnology & Informatics). Protocols: Protocol of seed germination:

1. Wash the seeds by submerging in the water along with few drops of detergent. Shake it or wrap the seeds in two layers of cloth and then wash it. 2. Submerge the seeds in 70% alcohol for 30 to 60 seconds. 3. Transfer the seed to flask containing 20 to 40% sodium hypochlorite for 15 to 20 minutes. Rinse 5 times with sterile distilled water. Place 2 or 3 seeds per culture vessel on the surface of MS agar medium without growth regulators. Incubate the culture at 25 degree under 16 hour photoperiod with approximately 100 lux light intensity for 1 to 2 weeks. Observe weekly for root initiation. Gently remove well-rooted plantlets form the culture vessel, keeping the roots intact. Plant the regenerates in small plastic pots with sterile soil, mix and make sure that soil is moist with water, then cover it with inverted plastic bag. Place the pots in the diffused light and daily remove the plastic bag to allow the air passage and gradually increase the time of plant air contact to make it used to the natural environmental conditions.
4. 5. 6. 7. 8. 9.
Protocol for embryo culture:

1. Harvest ears of maize, wheat or barley, after 12 to 14 days of crossing or pollination. Developed seeds should be in the milky stage. At this stage embryos are less than 1.5mm in diameter. 2. Place such seeds in a beaker and sterile them with 20% sodium hypochlorite solution for 10 minutes in laminar air flow rinse 3 to 5 times with sterile distilled water. 3. Place a sterilized grain on a sterilized slide and make cuts with needles above the embryo in the endosperm while holding the embryo stationery with the second needle. 4. Dissect out the immature embryo (1 to 1.5 mm diameter) from the seeds under a diseection microscope. 5. Incubate the cultures in light at 25%. 6. Transfer plant lets after 2 weeks to half strength MS medium or B-5 media. 7. Transfer plantlets to plastic pots and gradually expose them to the environmental conditions.
Protocol for embryo culture:

1. Collect developing pods in which the embryos are at heart stage and beginning of cotyledon stage. Depending on the species this stage will be approximately after 9 to 12 days of pollination. 2. Submerge the seeds in 70% alcohol for 30 to 60 seconds, then sterilize with 20% sodium hypochlorite solution for 10 minutes in laminar air flow hoods and then wash it 3 to 5 times with distilled water. 3. Place the pods on sterile slide and slit open the pods, and place the developing seeds on slide or Petri dish.

4. Remove the seed coat place directly above the embryo and carefully dissect out the embryos with forceps and needles. 5. Inoculate the embryo on MS medium. 6. Incubate the culture in light at 25 degree. 7. Transfer plantlets after 3 to 4 weeks on half strength MS media without growth regulators. 8. Transfer plantlets to plastic pots and gradually expose them to the environmental conditions.
Protocol for callus induction:

1. Follow the steps of isolation and sterilization. 2. Collect aseptically germinated seedlings when cotyledons are fully expanded and the epicotyl is beginning to emerge. Usually this will occur when the seedling are 2 weeks old. Place the seedling on sterile soils and prepare the ex-plant. 3. Place the ex-plant on MS media. 4. Incubate the culture in dark at 25 degree; callus will be produced in 3 to 4 weeks. 5. Compare the callus induction growth from various ex-plants. 6. Cut small pieces of callus and sub culture on the fresh medium for proliferation.
Protocol for callus induction (cereals- wheat, rice, maize, & barley etc)
1. Isolate the immature embryo and sterilize it. 2. Place 5 to 6 immature embryos per vessel following the media for callus induction(MS media +2-4 D). 3. Incubate the culture in dark at 25 degree; callus will be produced in 3 to 4 weeks. 4. Scutellar callus can be multiplied at least once on the same callus initiation medium by breaking into 2 to 3 pieces after 3 to 4 weeks of initiation of callus. 5. Place hard and complex pieces of callus of approximately 0.5 cm square on same callus initiation medium for proliferation.

BSTC-363 Cell &amp; Tissue Culture

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BSTC-363 Cell &amp; Tissue Culture

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Biotech Online. BS (Biotechnology & Informatics).

NOTES: Cell & Tissue Culture. BSTC-363 .