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The following points highlight the top six types of tissue culture. The types are: 1. Seed Culture 2. Embryo Culture 3. Callus Culture 4. Organ Culture 5. Protoplast Culture 6. Anther Culture.
Type # 1. Seed Culture:
Seed culture is an important technique when explants are taken from in vitro-derived plants and in propagation of orchids. Sterilising procedures are needed for plant materials that are to be used directly, as explant source can cause damage to tissues and affect regeneration. In that case, culture of seeds to raise sterile seedling is the best method. Orchid cloning in vivo is a very slow process.
Thus seeds can be germinated in vitro and vegetatively propagated by meristem culture is then carried out on a large scale. Most orchids are sown in vitro because: orchid seeds are very small and contain very little or no food reserves. Their small size (1.0-2.0 mm long and 0.5-1.0 mm wide) makes it very likely that they can be lost if sown in vivo, and the limited food reserves also make survival in vivo unlikely.
The seed consists of a thickened testa, enclosing an embryo of about 100 cells. The embryo has a round or spherical form. Most orchid seeds are not differentiated: there are no cotyledons, roots and/or endosperm.
The cells of an embryo have a simple structure and are poorly differentiated;
i) Sowing in vitro makes it possible to germinate immature orchid embryos, thus shortening the breeding cycle; and
ii) Germination and development take place much quicker in vitro since there is a conditioned environment and no competition with fungi or bacteria.
Orchid seeds imbibe water via the testa and become swollen. After cell division begun, the embryo cracks out of the seed coat. A protocorm-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 a shoot. Protocorms obtained by seed germination have many close similarities with those produced from isolated shoot tips; the term protocorm like-bodies has introduced when cloning orchids by meristem culture.
The vegetative propagation of orchids follows culture of seeds, transformation of meristem into protocorm-like bodies, and the propagation of protocorms by cutting them into pieces and the development of these protocorms to rooted shoots. A large number of factors influence the germination and growth of orchids. The mineral requirement of orchids is generally not high and a salt poor medium of Knudson and Vacin and Went are good.
Some of the orchids require (Paphiopedilum ciliolare) for germination while others requires low irradiance. Sugar is extremely important as an energy source, especially for those that germinate in darkness. Regulators are usually not necessary for seed germination, and their addition often leads to unwanted effects like callus formation, adventitious shoot formation, etc.
Type # 2. 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 embryos of angiosperms was made by Hannig in 1904 who obtained viable plants from in vitro isolated embryos of two crucifers Cochleria 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 organisation of a mature embryo.
They grew directly into seedlings, skipping the stages of normal embryogenesis and without the completion of dormancy period. Laibach demonstrated the practical application of this technique by isolating and growing the embryos of interspecific cross.
Linum perenne and L, austriacum that aborted in vivo. This led Laibach to suggest that in all crosses where viable seeds are not formed, it may be appropriate to exercise their embryos and grow them in an artificial nutrient medium.
There are two types of embryo culture:
i. 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. Seed dormancy of many species is due to chemical inhibitors or acids, mechanical resistance present in the structures covering the embryo, rather than dormancy of the embryonic tissue.
ii. Immature embryo culture/embryo rescue:
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 a viable plant. The underlying principle of embryo rescue technique is the aseptic isolation of embryo and its transfer to a suitable medium for development under optimum culture conditions.
Florets are removed at the proper time and either florets or ovaries are sterilised. Ovules can then be removed from the ovaries. The tissue within the ovule, in which the embryo is embedded, is already sterile.
For mature embryo culture either single mature seeds are disinfected or if the seeds are still unripe then the still closed fruit is disinfected. The embryos can then be aseptically removed from the ovules. Utilisation of embryo culture to overcome seed dormancy requires a different procedure.
Seeds that have hard coats are sterilised and soaked in water for few hours to few days. Sterile seeds are then split and the embryos excised. The most important aspect of embryo culture work is the selection of medium necessary to sustain continued growth of the embryo. In most cases a standard basal plant growth medium with major salts and trace elements may be utilised.
Mature embryos can be grown in a basal salt medium 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 a medium with high sucrose concentration (8-12%); which approximate the high osmotic potential of the intracellular environment of the young embryosac, and a combination of hormones which supports the growth of heart-stage embryos (a moderate level of auxin and a low level of cytokinin). Reduced organic nitrogen as aspargine, glutamine or casein hydrolysate is always beneficial for embryo culture.
Malic acid is often added to the embryo culture medium. After one or two weeks when embryo ceases to grow, it must be transferred, to a second medium with a normal sucrose concentration, low level of auxin and a moderate level of cytokinin which allows for renewed embryo growth with direct shoot germination in many cases.
In some cases where embryo does not show shoot formation directly, it can be transferred to a medium for callus induction followed by shoot induction. After the embryos have grown into plantlets in vitro, they are generally transferred to sterile soil and grown to maturity.
Applications of embryo culture are:
Prevention of embryo abortion in wide crosses: resignation
Successful interspecific hybrids have been seen in cotton, barley, tomato, rice, legume, flax and well known intergeneric hybrids include wheat x barley, wheat x rye, barley x rye, maize x Tripsacum, Raphanus sativus x Brassica napus.
Distant hybrids have also been obtained via embryo rescue in Carica and Citrus species. Embryo rescue technique has been successfully used for raising hybrid embryos between Actidinia deliciosa x A. eriantha and A. deliciosa x A. arguata.
Production of Haploids:
Embryo culture can be utilised in the production haploids or monoploids. Kasha and Kao (1970) have developed a technique to produce barley monoploids. Interspecific crosses are made with Horeum bulbosum as the pollen parent, and the resulting hybrid embryos are cultured but they exhibit H. bulbosum chromosome elimination resulting in monoploids of the female parent H. vulgare.
Overcoming seed dormancy:
Embryo culture technique is applied to break dormancy. Seed dormancy can be caused by numerous factors including endogenous inhibitors, specific light requirements, low temperature, storage requirements and embryo immaturity. These factors can be circumvented by embryo excision and culture.
Shortening of breeding cycle:
There are many species that exhibit seed dorm that is often localised in the seed coat and/or in the endosperm. By removing these inhibitions, seeds germinate immediately.
Seeds sometimes take up and O2 very slowly or not at all through the seed coat, and so germinate slowly if at all, e.g. Brussels sprouts, rose, apple, oil palm and iris. H (Ilex) is important plants for Christmas decorations. Ilex embryos remain in the immature heart-shaped stage though the fruits have reached maturity.
Prevention of embryo abortion with early ripening stone fruits:
Some species produce sterile seeds that will not germinate under appropriate conditions and eventually decay in soil e.g. early ripening varieties of peach, cherry, apple, plum. Seed sterility may be due to incomplete embryo development, which results in the death of the germinating embryo.
In crosses of early ripening stone fruits, the transport of water and nutrients to the yet immature is sometimes cut off too soon resulting in abortion of the embryo. Eg: Macapuno coconuts are priced for their characteristic soft endosperm which fills the whole nut.
These nuts always fail to germinate because the endosperm invariably rots before germinating embryo comes out of the shell. Embryo culture has been practised as a general method in horticultural crops includes avocado, peach, nectarine and plum. Two cultivars ‘Goldcrest peach’ and ‘Mayfire nectarine’ have resulted from embryo culture and commercially grown.
Embryos are excellent materials for in vitro clonal propagation:
This is especially true for conifers and members of Gramineae family.
Germination of seeds of obligatory parasites without the host is impossible in vivo, but is achievable with embryo culture.
Type # 3. Callus Culture:
Callus is basically a more or less non-organised tumor tissue which usually arises n wounds of differentiated tissues and organs. Thus, in principle, it is a non-organised and little differentiated tissue. The cells in callus are of a parenchymatous nature. When critically examined, callus culture is not homogeneous mass of cells, because it is usually made up of two types of tissue: differentiated and non- differentiated.
Explant tissue is a differentiated tissue (roots, stem, leaves, flowers, etc.) which is used as a starting material for callus induction. These explant tissues generally show distinct planes of cell division, cell proliferation and organisation into specialised structures such as vascular systems.
If there are only differentiated cells present in an isolated explant, then dedifferentiation must take place before II division can occur. Parenchyma cells present in the explant usually undergo is differentiation. If the explant already contains meristematic tissue when isolated, then this can divide immediately without dedifferentiation taking place.
Callus formation takes place under the influence of exogenously supplied growth regulators present in the nutrient medium. The type of growth regulator requirement and its concentration in the medium depends strongly on the genotype and endogenous hormone content of an explant.
These requirements can be put into three categories:
i. Auxin alone (especially in monocotyledons).
ii. Cytokinin alone
iii. Both auxin and cytokinin (carrot)
If the callus is difficult to induce, or if juvenile callus is needed, then immature embryos or seedlings or parts of these are used. It should be taken into account that type of starting material (juvenile or adult) and the original – position of the explant in the plant reflects the endogenous hormone level which has an important influence on processes such as cell division and organ and embryo formation. Many other factors are important for callus formation: genotype, composition the nutrient medium, physical growth factors (light, temperature, etc.).
Sucrose or glucose (2-4%) is usually employed as the sugar source. The effect light on callus formation is dependent on the plant species; light may be required in some cases and darkness in other cases. A temperature of 22-28°C is normally advantageous for callus formation.
After callus induction, the callus is grown further on a new medium which referred to as sub-culturing. When sub-cultured regularly on agar medium, callus cultures will exhibit an S shaped or sigmoid pattern of growth during each passage.
There are five phases of callus growth:
i. Lag phase, where cells prepare to divide.
ii. Exponential phase, where the rate of cell division is highest.
iii. Linear phase, where cell division slows but the rate of cell expansion increases.
iv. Deceleration phase, where the rates of cell division and elongation decreases.
v. Stationary phase, where the number and size of cells remain constant.
Callus growth can be monitored by fresh weight measurements, which convenient for observing the growth of cultures over time in a non-destructive manner. Dry weight measurements are more accurate than fresh weight, but method requires sacrifice of the samples. Mitotic index measurement of cell division rates require extensive sampling to reduce sample error and are not easy to perform.
Type # 4. Organ Culture:
It is an isolated organ grown in vitro. It can be given different names depends upon the organ used as an explant. For example, meristem or shoot tip culture, root culture, nucellus culture, endosperm culture, ovule culture, a culture for production of androgenic haploids while ovule and ovary culture vitro production of gynogenic haploids. The culture of plant results in three types of in vitro culture.
Organised:
The culture of whole plants (embryos, seeds) and organ has been termed as organised culture. In this, characteristic organised structure of a plants individual organ is maintained. If the organised structure is not broken down, progeny arise which are identical to the original plant material (e.g. meristem culture).
Non-organised:
If cells and/or tissues are isolated from an organised part of a plant, dedifferentiate and are then cultured, a non-organised growth in the form callus tissue results. If the callus disperses into clumps of cells (aggregates) a single cells result, it is referred to as suspension culture. Non-organised culture has very low genetic stability.
Non-organised/organised:
This type of culture is intermediate between the above two types. Cells in an isolated organ or tissue first dedifferentiate and then form tissues which then re-differentiate to form organs (roots or shoots) or embryos. Thus organised structures can develop from non-organised cultures either through techniques or spontaneously. In this the progeny are often not completely identical to the original plant material.
Type # 5. Protoplast Culture:
Now we will look at the different procedures of protoplast culture.
They are as follows:
Isolation of protoplasts:
Protoplasts (cell without cell wall) are the biologically active and most significant material of cell. Cooking for the first time isolated protoplasts of plant tissue by using cell wall degrading enzymes viz. cellulase, hemicellulase, pectinase, and protease extracted from a saprophytic fungus Trichoderma viride. Now the protoplasts are cultured in vitro. Sterilisation of the leaf samples with sodium hypochlorite solution.
Rinsing in suitable Osmaticum ie in distilled water or MS medium adjusted to a suitable pH and buffer to maintain osmotic pressure. Plasmolysis of cells takes place by keeping the stripped leaves in 13% mannitol for 3 hours. Peeled leaves are transferred into an already sterilised enzyme solution for 12-15 hours for the facilitation of the enzyme to enter the tissue.
Isolation and purification of protoplasts takes place by filtering the enzyme solution containing protoplasts through a nylon mesh (45pm). The filtrate is centrifuged 2-3 times repeating the above steps and finally a specific concentration of protoplast suspension is prepared.
Protoplast culture and regeneration:
From the protoplast solution of known density (about 105 protoplasts /ml) about 1ml suspension is poured on sterile and cooled down nutrient medium in Petri dishes. The plates are incubated at 25°C in a dim white light.
The protoplast regenerates a cell wall, undergo cell division and forms callus. The callus can be subcultured. Embryogenesis begins from callus when it is placed on nutrient medium lacking mannitol and auxin. The embryo develops into the seedling s and finally into mature plants.
Type # 6. Anther Culture:
Anther culture is the process of using anthers to culture haploid plantlets. The technique was discovered in 1964 by Guha and Maheshwari. This technique can be used in over 200 species, including tomato, rice, tobacco, barley, and geranium.
Some of the advantages which make this a valuable method for obtaining haploid plants are: the technique is fairly simple it is easy to induce cell division in the immature pollen cells in some species a large proportion of the anthers used in culture respond (induction frequency is high) haploids can be produced in large numbers very quickly.
In experiments using Datura innoxia, induction frequencies of almost 100% and a yield of more than one thousand plantlets or calluses have occurred under optimal conditions from one anther. Success can be determined within 24 hours as cells begin to divide.