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A research paper on plant tissue culture. This research paper will help you to learn about: 1. Meaning of Plant Tissue Culture 2. Nutrient Medium for Plant Tissue Culture 3. Aseptic Conditions 4. Aeration 5. Cellular Totipotency 6. Applications 7. Culture of Plant Materials 8. Regeneration of Plantlets 9. Anther Culture and Haploid Production 10. Protoplast Culture and Somatic Hybridisation Other Details.
Contents:
- Research Paper on the Meaning of Plant Tissue Culture
- Research Paper on Nutrient Medium for Plant Tissue Culture
- Research Paper on Aseptic Conditions for Plant Tissue Culture
- Research Paper on Aeration Required for Plant Tissue Culture
- Research Paper on Cellular Totipotency helps in Plant Tissue Culture
- Research Paper on the Applications of Plant Tissue Culture
- Research Paper on the Culture of Plant Materials in Plant Tissue Culture
- Research Paper on the Regeneration of Plantlets in Plant Tissue Culture
- Research Paper on Anther Culture and Haploid Production of Plant Tissue Culture
- Research Paper on Protoplast Culture and Somatic Hybridisation of Plant Tissue Culture
- Research Paper on the Culture of Shoot Apices and Leaves of Plant Tissue Culture
- Research Paper on the Culture of Ovaries
- Research Paper on the Culture of Endosperms
Research Paper # 1. Meaning of Plant Tissue Culture:
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Plant tissue culture is the maintenance and growth of plant cells, tissues and organs on a suitable culture medium in vitro, e.g., in a test tube or any other suitable vessel. Plant tissue cultures are often classified according to the type of in vitro growth, such as callus and suspension cultures, or the explants used for culture initiation, e.g. embryo culture, another culture, etc.
An explants is the part of a plant that is excised from its original location and used for initiating a culture.
Surface sterilisation and sterilisation. It is essential that the explants, glassware, culture containers or vessels, media and the instruments used for plant tissue culture must be free from microbes. Hence the explants are treated with specific anti-microbial chemicals, and the process is called surface sterilisation.
Suitable sized plant material (explant) is sterilised as follows:
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(i) Bring the explant to be sterilised in well sterilised laboratory and prepare pieces for sterilisation.
(ii) Clean the working area and hands with alcohol, put on mask and cap, and light the spirit lamp.
(iii) Keep 3 or 4 petridishes in a line, add disinfectant (e.g., mecuric chloride 0.01 to 0.1% aqueous solution, or 20% sodium hydrochloride) in first plate and autoclaved distilled water in subsequent plates.
(iv) Place plant pieces in first plate and immerse the material with the help of sterilised forceps for 5-10 minutes depending upon the disinfectant used.
(v) Transfer material from first to second petriplate, rinse gently and pass to third and fourth plates, one by one with thorough rinsing.
(vi) Finally drain the distilled water, and prepare suitable sized explants.
A quick dip in 70% ethanol (15-30 seconds), is always advantageous, before surface sterilisation with disinfectant.
The vessels, media and instruments are also suitably treated with steam, dry heat, alcohol or subjected to filtration to make them free from microbes.
Generally, autoclave is used to sterilise medium, glassware and tools for the purpose of plant tissue culture. Sterilisation of material is carried out by increasing moist heat (121°C) due to increased pressure inside the vessel (15-22 psi, i.e., pounds per square inch) for 15 minutes for routine sterilisation. Moist heat kills the microorganisms and makes the material free from microbes.
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Surface strerilisation of explants and their transfer to culture media must be done under aseptic conditions.
Nutrient media could be prepared in a separate room where sufficient space is available for keeping and weighing chemicals, and putting glassware.
Vitamins and growth hormones are carefully weighed.
Stock solutions of chemicals are kept in refrigerator to avoid contamination.
The technique of plant tissue culture enables us to study the cells, tissue or organs by isolating them from the plant body and growing aseptically, in suitable containers, on an artificial nutrient medium, under controlled environmental conditions.
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Thus (i) Nutrient medium (ii) Aseptic conditions and (iii) Aeration of the tissue are important aspects of the technique of in vitro culture.
Research Paper # 2. Nutrient Medium for Plant Tissue Culture:
Every tissue and organ has its special requirements for optimal growth and these needs to be worked out when starting work with a new system (Table 9.1). However, most of the media contain inorganic salts of major and minor elements, vitamins and sucrose.
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A medium with these ingredients will be referred to as basal medium, sometimes, growth regulators, such as auxins, gibberellins and cytokinins, may also be added to the basal medium. Growth regulators are required for cell division and organ regeneration from the cultures.
The cultures are usually kept in a culture room at about 24°C with some illumination. These all constituents are dissolved in distilled water. If necessary, the medium is solidified with about 0.8% agar. The pH of the medium is adjusted around 5.8 (slightly acidic).
Now equal quantities of the medium are dispersed in culture vials, which are usually glass tubes or flasks. The culture vials, containing medium, are plugged with non-absorbent cotton wrapped in cheese cloth. Such a closure allows the exchange of gases but does not permit the entry of micro-organisms into culture vials.
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Research Paper # 3. Aseptic Conditions for Plant Tissue Culture:
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The sugar content of the nutrient media may support a luxuriant growth of many microorganisms, like bacteria and fungi. It is, therefore, extremely important to maintain a completely aseptic environment inside the culture vials.
Micro-organisms can contaminate the medium in at least three ways:
(a) The micro-organisms present in the medium right from the beginning many be destroyed by sterilizing the properly plugged culture vials. It can be done by maintaining the temperature at 120°C for about 15 minutes.
(b) The micro-organisms may also be carried along with tissue that is being cultured. To prevent this, the plant material from which the tissue is to be excised is surface sterilized. The material may be surface sterilized with saturated chlorine water and then thoroughly washed with sterilized distilled water and to remove all traces of chlorine. If the material is fairly hard, as are some fruits and seeds, it may be surface sterilized by rinsing in alcohol.
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(c) Finally, precautions must also be taken to prevent the entry of micro-organisms while the plug of a culture vial is removed to transfer the tissue to the nutrient medium (inoculation) For this, all operations from surface sterilization of the tissue up to inoculation are done in an aseptic environment.
Research Paper # 4. Aeration Required for Plant Tissue Culture:
Proper aeration of the cultured tissue is also an important aspect of culture technique. If the tissue IS grown on the surface of a semi-solid medium it acquires enough aeration without special device for liquid medium, special device “filter paper bridge” is used. Here, two legs remain medium.
Research Paper # 5. Cellular Totipotency helps in Plant Tissue Culture:
This is the capacity of nature cells showing that when freed from the plant body, they had the ability to reorganize themselves into the new individuals. Steward and his co-workers showed this phenomenon in the carrot cultures. Here the small pieces of mature carrot root were grown in a liquid medium supplemented with coconut milk, in special containers.
These cultures were shaken generally which freed all the cell clusters into the medium Some developed into rooting clumps When these were transferred to the tubes containing a semi-solid medium, they gave rise to whole plant that flowered and set seeds. On the basis of these experiments, it can be inferred that, at least theoretically, every living plant cell, irrespective of its age and location is totipotent.
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However, this phenomenon cannot be compared with the mode of the development of the zygote, wherein the divisions give predictable manner. But in case of cultured cells, the isolated single cells of tobacco divide, quite irregularly to form a mass from which roots and shoot buds differentiate eventually.
However, Guha and Maheswari, while culturing mature anthers of Datura innoxia with an aim of understanding the physiology of meiosis, accidentally noticed that on basal nutrient medium containing kinetin, coconut milk, or grape juice numerous embryo-like structure appeared from the inside of anthers, which eventually developed into plants.
Later on these workers confirmed the origin of the embryoids from pollen gram. As expected, the plantlets of pollen-origin were haploid.
Research Paper # 6. Applications of Plant Tissue Culture:
(a) The technique provides a way for rapid multiplication of desirable and rare plants.
(b) As the experiments reveal, we may obtain healthy stocks from virus infected plants through shoot-tip culture.
(c) The development of haploids through the techniques of another culture is having its potential significance in basic and applied genetics and plant breeding. During the Past 30 years, the technique has been successfully extended to about 20 species including some economic plants, e.g., Atropa, Brassica, Hordeum, Lycopersicon, Nicotiana. Oryza and Triticum.
(d) The embryo culture has been useful in overcoming seed dormancy. It is also utilized for producing viable plants from crosses which normally fail due to the death of immature embryos, e.g… Jute, rice.
(e) The embryo tissue culture is also applied for the propagation of rare Plants, e.g.. “makapuno”. Here, some coconuts develop soft, solid, fatty tissue in Place of the liquid endosperm.
These are rare and very expensive, served only at special banquets in the Philippines. Under normal conditions the coconut seeds fail to germinate. Using the technique of in vitro culture of excised embryos, the scientists have succeeded in plantlets from makapuno nuts.
(f) Another important use of embryo culture is in obtaining some rare hybrids. It is possible to raise complete hybrid plants through embryo culture. This method has been profitably used for many interspecific crosses.
Research Paper # 7. Culture of Plant Materials in Plant Tissue Culture:
Explant Culture:
There are a variety of forms of seed plants, such as trees, herbs grass, which exhibit the basic morphological units, i.e., root, stem and leaves, versatile of all types of tissues. They are capable of division and growth.
Development of a tissue is characterized:
(i) Cell division,
(ii) Cell elongation, and
(iii) Cell differentiation.
For this reason, the explants from healthy and young part of the plant are used. Presence of parenchyma is first consideration in a particular species, parenchyma from stems, rhizomes, tubers; root is easily accessible and will generally respond quickly to culture conditions in vitro.
Callus Formation and its Culture:
In nature, callus develops by infection of microorganisms from wounds due to stimulation by endogenous growth hormones, the auxins and cytokinins. However, it has been artificially developed by adopting tissue culture techniques.
A callus is an amorphous mass of loosely arranged thin walled parenchyma cells developing from proliferating cells of the parent tissue. The unique feature of callus is that the abnormal growth has logical potential to develop normal root, shoots and embryoids ultimately forming a plant.
In callus culture, cell division in the explant forms a callus, an un-organised mass of cells. It is maintained on a medium gelled usually with agar. The medium ordinarily contains the auxin 2, 4 – D (2, 4 – dichlorophenoxyacetic acid), and often a cytokinin BAP (benzylaminopurine).
When an explants is placed on such a medium, many of the cells become meristematic and begin to divide. In about 2 to 3 weeks, a callus mass is obtained.
Cell (Suspension) Culture:
A suspension culture consists of single cells and small groups of cells suspended in liquid medium. Cell suspension is prepared by transferring a fragment of callus (about 500 mg.) to the liquid medium (500 ml.) and agitating them aseptically to make the cells free in medium. The medium ordinarily contains the auxin 2, 4 – D. Suspension cultures must be constantly agitated at 100 – 250 rpm (revolutions per minute).
Agitation serves three important purposes.
They are as follows:
(i) Aeration of culture;
(ii) Constant mixing of the medium and
(iii) Breakage of cell aggregates into smaller cell groups. Suspension cultures grow much faster than callus cultures.
It is difficult to have suspension of single cell. However, the suspension includes single cell, cell aggregates (varied number of cells), residual inoculum and dead cells. King (1980) has described that a good suspension consists of a high proportion of single cells than small cluster of cells.
Cell suspension cultures have many advantages over the callus cultures.
They are as follows:
(i) The suspension can be pipetted.
(ii) They are less heterogeneous and cell differentiation is less pronounced.
(iii) They can be cultured in volumes up-to 1500 litres.
(iv) They can be subjected to more stringent environmental controls.
(v) They can be manipulated for production of natural products by feeding precursors.
Sub-Culturing:
After some time, the under-mentioned three things happen in all types of plant tissue cultures:
(i) Cell/tissue dry matter known as biomass increases,
(ii) The level of nutrients in the medium decreases, and
(iii) The medium volume declines due to evaporation.
Hence, if tissue cultures were kept in the same culture vessel, they will die in due course of time. Due to this reason, cells/tissues are regularly transferred into new culture vessels containing fresh media. This process is called sub-culturing.
Precaution is taken that during sub-culture, only a part of the culture from a vessel is transferred into the new culture vessel.
Difference between callus and suspension cultures:
Callus Culture:
a. Her cell division in the explant forms a callus, which is an unorganised mass of cells.
b. It is maintained on a medium gelled usually with agar.
c. The medium ordinarily contain the auxin 2, 4-D, and often a cytokinin like BAP.
d. There is no need of agitation.
e. In about 2-3 weeks, a callus mass is obtained.
Suspension Culture:
a. Suspension culture consists of single cells and small groups of cells.
b. Here, cells are suspended in liquid medium.
c. Usually the medium contains the auxin 2, 4-D.
d. They must be constantly agitated at 100-250 rpm (revolutions per minute).
e. suspension culture grow much faster than callus cultures.
Uses of Callus and Suspension Cultures:
The callus and suspension cultures can be used to achieve cell biomass production which may be used for biochemical isolation.
Also used for regeneration of plantlets, i.e., newly regenerated plants through tissue culture.
Production of transgenic plants and isolation of protoplasts
Research Paper # 8. Regeneration of Plantlets in Plant Tissue Culture:
Plants cells cultured in vitro can ultimately give rise to complete plants. Potentiality of a plant cell to regenerate the entire organism or plant is called totipotency.
This potentiality has been exploited through the culture of protoplasts, cells, tissues and organs in vitro. Gottlieb Haberlandt in 1902, for the first time started the technique of plant tissue culture, when he attempted to culture isolated single cells from leaf mesophyll to determine their totipotency.
Regeneration describes the development of an organised structure, such as root, shoot or somatic embryo from cultured cells.
However, plantlets can be obtained from cultured cells by two different ways:
(i) Where shoot regeneration is followed by rooting of the shoots and
(ii) Where regeneration of somatic embryos followed by their germination.
In cultured materials it has been possible to study such process as differentiation of a parenchyma cell into tracheid (i.e., cytodifferentiation), organ formation (i.e., organogenesis) and somatic embryo formation (i.e., somatic embryogenesis). Besides the study of fundamental process of differentiation, the capacity of cell to form organs and embryos can be exploited to regenerate plantlets for clonal propagation.
Organogenesis:
The process by which cells and tissues are forced to undergo changes which lead to the production of a structure, known as shoot or root primordium. This system is commonly produced in callus cultures. The earliest reports on controlled organogenesis in vitro were by White (1939) who obtained shoots on callus of a tobacco hybrid and by Nobecourt (1939), who observed root formation in carrot callus.
The earliest report on controlled somatic embryogenesis in vitro was with carrot reported simultaneously in 1958 by Reinert and Steward (see Fig 9.3). The process occurs naturally in a wide range of species from both reproductive and somatic tissues. Somatic embryos can be formed on callus, in cell suspensions and protoplasts cultures, or directly from cells of organised structures, such as stem segments or zygotic embryo.
Advantages of Plantlet Regeneration:
There are several advantages of plantlet regeneration through plant biotechnological methods using organogenesis or embryogenesis, in comparison to conventional methods of propagation.
The advantages include the efficiency of process, i.e., formation of plantlet in fewer steps, with reduction in labour, time and cost, and the potential for the production of much higher number of plantlets, and their morphological and cytological uniformity. Till now about 150 species from angiosperms and gymnosperms have been reported to produce somatic embryos in culture.
Shoot and Root Regeneration:
Shoot regeneration is promoted by a cytokinin, such as BAP (benzylaminopurine). While root regeneration is promoted by an auxin, such as NAA (naphthalene acetic acid). Thus, shoot and root regenerations are generally controlled by auxin- cytokinin balance. Usually, an excess of auxin promotes root generation, and that of cytokinin promotes shoot generation.
Callus cultures are first kept on a BAP (cytokinin) containing medium. After sometime shoots regenerate from callus cells. When the shoots become 2-3 cm long, they are excised and transferred to an auxin-containing medium. After sometime, roots regenerate from the lower ends of these shoots to give rise to complete plantlets.
Somatic Embryo Regeneration:
Plant cells are totipotent and can produce complete new plants under favourable conditions of nutrients and plant growth regulators. Steward and Reneirt, almost simultaneously reported for the first time, somatic embryo formation in carrot cell suspension cultures in 1958. These somatic embryos were similar to zygotic embryos in development and structure.
A somatic embryo develops from a somatic cell. The pattern of development of a somatic embryo is similar to that of a zygotic embryo.
Somatic embryo regeneration is induced usually by a relatively high concentration of an auxin like 2, 4-D (2, 4-dichorophenoxyacetic acid). The young embryos develop into mature embryos either in the same medium or on another medium. Mature somatic embryos germinate to yield complete plantlets.
During somatic embryo generation in cell suspension cultures, embryos of different sizes are produced. For any experimental or micro-propagation method, embryos of uniform size are required. This can be achieved by sieving or fractionation of suspension with appropriate sieve size.
Somatic embryo regeneration is a versatile technique for micro-propagation of plant species. A large number of herbaceous dicots and monocots have been regenerated through somatic embryogenesis.
Establishment of Plantlets in the Field:
Plantlets are produced through rooting of isolated shoots or germination of somatic embryos. Now, the plantlets can be removed from culture vessels and established in the field. This stage is concerned with transfer of plantlets in pots, their hardening and establishment in soil. Hardening of plants imparts some tolerance to moisture stress and plants become autotrophic from heterotrophic condition.
During hardening, plantlets are kept under a reduced light and high humidity for a suitable period of time. Hardening procedures make the plantlets capable of tolerating the relatively harsher environments outside the culture vessels.
Hardened plants are then transferred to glass or poly-houses with normal environmental conditions. Generally the poly-houses are erected by mounting polythene or polycarbonate sheets on metal frame support. Now, plants are irrigated frequently and their growth and variation monitored regularly. Plants are generally transferred to fields, e.g., plantation crops, after 4-6 weeks of acclimatization.
In India, there are now many commercial companies which produce millions of plantlets through micro-propagation.
Large glass houses and green houses are essential components of micro-propagation industry. Hardening and acclimatization of delicate in vitro raised plantlets is carried out in these glass houses.
Now-a-days chambers made of polycarbonate and polypropylene sheets are used for creating large working place. These houses are fitted with mist and fog generating units with cyclic auto-regulation. Light is provided through proper light sources.
Application:
Tissue culture has been successfully employed for the multiplication of orchids and many other ornamental plants.
In the developed countries tissue culture is a routine method of multiplication while in developing countries, such as India, the techniques are largely used for producing plants for export markets.
With tree species, the technique of tissue culture remained restricted for many years to the laboratory stage and has generally invited only academic interest.
To bridge this gap between research and application, the Department of Biotechnology (DBT), Government of India, set two pilot-scale national facilities for large-scale production of elite planting material of forest trees through tissue culture, one at the Tata Energy Research Institute (TERI), New Delhi, and the other at the National Chemical Laboratory (NCL), Pune.
However, practical applications of plant tissue culture are mainly based on the ability of plant cells to give rise to complete plantlets. The use of plant cells to generate useful products is called plant biotechnology. In most of its activities, the useful product is plantlet that, in many cases may have been genetically altered.
Usually, these plantlets are used for the following important purposes:
(i) Clonal Propagation:
Clonal propagation by vegetative methods is a practice followed since man started cultivation of plants. The main objective of clonal propagation has been to reproduce plants of selected desirable qualities uniformly and in bulk. The traditional propagation methods, require long duration, whereas tissue culture helps in rapid plant multiplication.
A clone is a group of individuals or cells derived from a single parent individual or cell through an asexual reproduction. All the cells in callus or suspension culture are derived from a single explants by mitotic division.
Hence, all plantlets regenerated from a callus or suspension culture generally have the same genotype and constitute a clone.
These plantlets can be used for rapid clonal propagation of superior lines.
Selected examples of clonal multiplication of trees and horticulture plants are as follows:
Oil palm, citrus peach, prunus, poplar, etc. Improved cultivars developed by biotechnological methods are then clonally multiplied to replace inferior cultivated varieties, e.g., in Mentha and other aromatic plants.
(ii) Somaclonal Variation:
Genetic variation present among plant cells of a culture is called somaclonal variation.
In vitro cell and tissue cultures of plant give rise to genetic variation spontaneously. While spontaneous variation is not desired during propagule multiplication, it has been useful in providing some genetic variants among crop species. The observed frequencies of such somaclonal variants vary widely and are probably related to both time and culture.
Several point mutations have been observed; the majority of analysed spontaneous genetic variants from somatic cultures appear to have resulted from induction of aneuploidy, loss of interchanges or intra-chromosomal segment duplication, deletions or structural arrangements.
Spontaneous or mutagen induced genetic variation in somatic cell culture coupled with in vitro selection techniques have been effective in isolating desired novel genetic variants with cellular level expression while in heterozygous condition. The somaclonal variation has been used to develop several useful varieties of crop plants.
(iii) Transgenic Plants:
A gene that is transferred into an organism by genetic engineering is called transgene. An organism that contains and expresses a transgene is called transgenic organism.
The plants, in which a functional foreign gene has been incorporated by any biotechnological methods that generally not present in plant, are called transgenic plants. However, a number of transgenic plants carrying genes for traits of economic importance have either been released for commercial cultivation or are under field trials.
However, transgenes can be introduced into individual plant cells. The cells containing and expressing transgenes can be easily selected in vitro. Ultimately, plantlets can be regenerated from these cells. These plantlets yield highly valuable transgenic plants.
Some of the commercially grown transgenic crop plants in developed countries are: ‘Flavr Savri’ and ‘Endless Summer’ tomatoes, ‘Freedom IP squash, ‘High lauric’ rapeseed and ‘Roundup Ready’ soybean. So far more than 60 transgenic dicot plants including herbs, shrubs and trees, and several monocots, such as maize, oat, rice, wheat, etc., have been produced.
In future the number of these crops certainly will go up. These transgenic plants contain certain selected traits such as herbicide resistance, insect resistance, virus resistance, seed storage protein, modified ripening, modified seed oil, agglutinin, etc.
Meristem Culture:
The meristem is a dome of actively dividing cells about 0.1 mm in diameter and 0.25 mm in length. Shoots of all flowering plants grow by virtue of their apical meristems. The totipotency of the plant cells forms the basis of meristem culture.
Here, one can use an explant containing pre-existing shoot meristems, and produce shoots from them. Such, cultures are known as meristem culture. The explants generally used in meristem culture are shoot tips or nodal segments.
These explants may be cultured on a medium containing a cytokinin, usually BAP (benzylaminopurine). Cytokinins promote axillary branching by overcoming apical dominance. Hence, they support multiple shoot development from each explant.
When axillary branching takes place, individual shoots are cultured, whereas when axillary branching does not take place, the single shoot is cut into nodal segments, which are then cultured. Shoots of 2-3 cm are excised and rooted on a suitable medium.
The in vitro culture of meristem and shoot tip involves several phases such as initiation of culture and establishment of explant, growth and differentiation, proliferation of shoots and finally plantlet fomation by rooting of shoots.
The plantlets thus obtained are subjected to hardening and ultimately established in the field.
Uses of Meristem Cultures:
Meristem cultures are used for rapid clonal multiplication. Development of virus free plants is one of the most significant application of meristem culture. Generally this technique has applications in diverse areas such as clonal multiplication of vegetatively propagated crop plants, virus elimination and germplasm preservation.
Embryo Culture:
Interspecific crosses may fail due to several reasons, but when the development of embryo is arrested owing to the degeneration of the endosperm, or when the embryo aborts at an early stage of development, embryo culture is the only technique to recover hybrid plants.
It is being used extensively in the extraction of haploid barley (Hordeum vulgare) from the crosses H. vulgare x H. bulbosum. Embryo culture is also a routine technique employed in orchid propagation and inbreeding of those species that show dormancy. Das and Burman (1992) developed the method of regeneration of tea shoots from embryo callus.
Excision of young embryos from developing seeds and their cultivation on a nutrient medium is called embryo culture. In vitro older embryos are more easily cultured than young embryos.
The objective of embryo culture is to allow the young embryos to complete development and ultimately, give rise to seedlings.
More recently, a number of hybrids have been successfully raised through embryo culture: Hordeum vulgare × Secale cereale, H. vulgare × Agropyron repens, H. vulgare × Triticum aestivum, interspecific hybrids in Abelmoschus and Secale.
In some interspecific crosses, the endosperm of developing hybrid seeds degenerates at an early stage. Here, young embryos also die on the degeneration of endosperm. Hence such interspecific crosses cannot be normally made. In such cases, young hybrid embryos are excised and cultured in vitro to obtain hybrid seedlings.
The orchid industry owes a great deal to the technique of embryo culture. The culture of orchid embryos was initiated at the Singapore Botanic Garden in 1928. Orchids lack stored food. Here, embryo culture allows development of seedlings from most of the embryos. In such cases, embryo culture is also used for rapid clonal propagation.
Embryo culture has helped in overcoming self-sterility of seeds, especially of crop plants propagated vegetatively, when the seeds do not germinate in nature. In a wild relative of commercial banana, Musa balbisiana, for example, the seeds do not germinate under natural conditions. However, if the embryos are excised and grown on a simple culture medium of mineral salts, seedlings are readily obtained.
Indian Work:
Sometimes some interspecific hybrids of Oryza show quite a low percentage of seed germination under normal conditions because the hybrid embryos succumb at an early stage of their development. The scientists raised seedlings of certain interspecific crosses in Oryza by culturing the excised embryos on Nitsch’s medium .
The embryo culture technique has also been successfully employed in Colocasia esculenta where the seeds fail to germinate in nature. They grew the excised embroys on synthetic media and obtained seedlings which on transplanting in soil gave rise to normal healthy plants.
Rangaswamy and Rangan (1963) have cultured the embroys of a stem parasite, Cassytha filiformis, in the absence of the host, on a modified White’s medium supplemented with lAA. The embryos of Cuscuta reflexa, a total stem parasite, were also cultured.
Somewhat similar results have been obtained with the embryos of Dendrophthoe falcata, a partial stem parasite. Like the embryos of stem parasites, those of some root parasites have also shown considerable capacity for proliferation.
In cultures of seeds of Orobanche aegyptiaca, a total root parasite, the ovoid unorganized embryos produced a massive callus capable of continuous growth followed by differentiation into shoot tips.
Research Paper # 9. Anther Culture and Haploid Production in Plant Tissue Culture:
Anther, a male reproductive organ, is diploid (2n) in chromosome numbers. As a result of microsporogenesis, tetrads of microspores or pollen grains are formed from a single spore mother cell (2n), which are haploid (n).
In nature, haploid plants originate from unfertilized egg cells. However, in laboratory, they can be produced from both male and female gametes. In many plant species, haploids are produced when their anthers are cultured on a suitable medium. This process is called anther culture.
The first successful pollen cultures were established by Tulecke in the 1950’s using mature pollen grains of certain gymnosperms. In 1964, Guha and Maheshwari discovered that when excised anthers of Datura innoxia were cultured intact in a mineral salt medium in conjunction with coconut milk and other complex organic substances and growth hormones, embryo-like outgrowths appeared from the sides of the anther in about 6-7 weeks. In subsequent studies, they confirmed the haploid nature of the embryoids and their origin from microspores.
Investigations have shown that for some reason, anthers from flowers of the Solanaceae respond best to excision and culture by production of embryoids. Convincing demonstration of the direct transformation of microspores into embryoids has been provided by several workers in different species of the genera of Solanaceae, i.e.. Datura, Nicotiana, Atropa, Lycium, Petunia, Solanum, Capsicum and Hyoscyamus.
It has been observed that embryoids go through the globular, heart-shaped and torpedo stages typical of the ontogeny of normal diploid zygotic embryos before they finally elongate and form shoot and root meristems.
Embryoids have also been obtained from cultured anthers of certain cereals like rice. All in all therefore, the capacity of microspores in cultured anthers to form haploid embryoids and plants can now scarcely be questioned.
In some other plants, haploids do not arise directly from the microspores, but do through the intervention of a callus. The microspores first develop into multicellular bodies which later give rise to an exceedingly dense callus. Subculture of the callus in an embryogenesis-inducing medium containing specific concentration of auxins and a cytokinin led to the initiation of haploid seedlings.
There, thus seem to exist the following alternative pathways for haploidy in anther cultures:
a. Microspore → callus → embryogenesisà plantlet e.g., Solanaceae
b. Microspore → callus à Embryogenesis à Plantlet e.g., Oryza, Brassica, Hordeum, Coffea, Populus.
c. Microspore à callus à plantlet, e.g.. Datura metelloides.
In several species, the pollen grains can be isolated and cultured to obtain haploids. In many species of plants, haploids can also be produced by culturing unfertilised ovaries or ovules.
Applications:
Haploids, such produced are completely sterile and of no direct value But they are important as they are used to produce homozygous lines in 2-3 years. This strategy can be of much value in breeding programmes. Anthers from F1, plants, obtained by crossing of or more lines are cultured to obtain haploid plants.
The chromosome number of haploid plants may be doubled by using colchicine to obtain homozygous plants. The progeny from these plants are the subjected to selection to isolate superior homozygous lines.
Thus, haploid plants are very useful in (i) direct screening of recessive mutation in diploid ore polyploidy, screening of recessive mutation is not possible, and (ii) development of homozygous diploid plants following chromosome doubling of haploid plant cells.
In China the most widely grown wheat is a doubled haploid produced through homozygous diploid lines. Another culture of rice is also successfully grown. Haploid plants have been produced in tobacco, wheat and rice through pollen culture which are used for the development of disease resistant and superior diploid lines.
At present more than 247 plant species and hybrids belonging to 38 genera and 34 families of dicots and monocots have been regenerated using anther culture technique. They include economically important crops and trees, such as rice, wheat, maize, coconut, rubber plantation, etc.
Research Paper # 10. Protoplast Culture and Somatic Hybridisation in Plant Tissue Culture:
An even more advanced technique of free cell culture is that involving the culture of isolated protoplasts. Essentially, the method consists of, first, breaking down the cell wall mechanically or chemically using enzymes and thus freeing the protoplasts which are then cultured like whole cells using appropriate culture media. The cells are, in other words, rendered naked and then cultured.
When cell wall is mechanically or enzymatically removed, the isolated protoplast is known as ‘naked plant cell’ on which most of recent researches are based.
Plant cell wall acts as physical barrier and protects cytoplasm from microbial invasion and environmental stress. Cooking (1960), for the first time isolated the protoplasts of plant tissues by using cell wall degrading enzymes, viz., cellulose, hemicellulose, pectinase and protease extracted from a saprophytic fungus Trichoderma viride. Later on protoplasts were cultured in vitro.
Protoplast Culture and Regeneration:
The protoplasts regenerate a cell wall, undergo cell division and form callus. The callus can also be sub-cultured. Embryogenesis begins from callus when it is placed on nutrient medium lacking mannitol and auxin. The embryo develops into seedlings and finally mature plants.
Somatic Hybridization:
Fusion between protoplasts of the selected parents is induced by a solution of polyethylene glycol (PEG), or by very brief high voltage electric current. Somatic hybridisation allows the production of hybrids between lines and species that cannot be produced normally by means of sexual hybridisation.
Fusion of cytoplasm of two protoplasts results in coalescence of cytoplasm. The nuclei of two protoplasts may or may not fuse together even after fusion of cytoplasms. The binueleate cells are known as heterokaryon.
When nuclei are fused the cells are known as hybrid, and when only cytoplasms fuse and genetic information from one of the two nuclei is lost is known as cybride i.e, cytoplasmic fuse and genetic information from one of the two nuclei is lost is know as cybrid i.e, cytoplasmic hybrid.
However, production of cybrids which contain the mixture of cytoplasms but only one nuclear genome can help in transfer of cytoplasmic genetic information from one plant to another. Thus, information of cybrid can be applicable in plant breeding experiments.
For example, in China, cybrid technology in rice is a great success. Such plants are very useful in producing hybrid seeds without emasculation. Today, cybrid technology has successfully been applied to carrot, mustard, citnis, tobacco and sugar beet.
Somatic hybrids may also be used for gene transfer, transfer of cytoplasm and production of useful allopolypoloids.
Tissue and protoplast cultures have been used in genetic engineering for the transfer of DNA and extrachromosomal bodies — plasmids, mitochondria, chloroplasts, nif (nitrogen fixing) genes from the nitrogen fixing bacterium Klebsiella pneumoniae to a strain of the colon bacterium Escherichia coli. Isolated protoplasts have great advantage in all the afore mentioned uses.
For transformation purposes cultured apical meristems are also usable because these can easily be regenerated into whole plants and also because intact DNA taken up by plants appears to be rapidly transported to meristematic regions, where growth and differentiation are centred.
Root Culture:
Excised roots were the first plant organs of higher plants to be successfully brought into sterile culture. In addition to the usual requirements for a carbon source and mineral nutrients most isolated roots grown in sterile culture required to be supplied with certain vitamins.
This is because, in the intact plant, certain vitamins are synthesized in the leaves and the roots are dependent upon shoots for the supply of these substances which they are unable to synthesize themselves.
For example, tomato roots require only sucrose, mineral nutrients and thiamin, and given all these, will grow successfully in culture for many years. But excised roots of some monocotyledonous plants fail to grow even when supplied with a full complement of β-vitamins and other vitamins.
In some of these (e.g., rye), an exogenous auxin supplement to the nutrient medium allows growth to proceed. In order to maintain the culture, the excised roots must be regularly sub-cultured on to a fresh medium, by excising a piece of root bearing a lateral which then proceeds to grow rapidly and maintain the culture.
As a rule, excised root of most species produce only root tissues in culture. There are exceptions, however, where they regenerate shoot buds as well as further roots, e.g.. Convolvulus. Taraxacum and Rumex.
Generally excised root is cultured in liquid medium. The techniques of root culture give certain important informations such as, (i) nutritional requirements, (ii) infection by Rhizobium and nodulation and (iii) physiological activities.
Research Paper # 11. Culture of Shoot Apices and Leaves of Plant Tissue Culture:
Like roots, isolated shoot apical meristems and leaf primordia can also be grown in sterile culture. These are often preferred in fact, because these frequently produce roots and can hence eventually develop into complete plants. The shoot apices and leaves of vascular cryptogams, such as ferns, are relatively more autotrophic than those of angiosperms.
Thus, even a small fern apex can be grown on a medium containing only carbohydrate source and mineral nutrients. Small angiosperm apices (less than 0.5 mm in diameter) require a general source of organic nitrogen and certain specific amino acids and vitamins in addition to the basic medium, but larger apices will grow on a simple medium.
The simpler requirements of larger apices may be due to the fact that they carry larger leaf primordia, which apparently can supply some of the requirements of the apex for vitamins and other organic nutrients.
Isolated young leaves of the fern Osmunda cinnamomea and of the sunflower (Helianthus annuus) and tobacco (Nicotiana tabacum) have been successfully grown on a simple medium containing only sucrose and inorganic salts.
Such isolated leaf primordia continue to grow and develop into normally differentiated leaves, although the latter are usually very much smaller than normal leaves developed in vivo, i.e., on the plant.
Research Paper # 12. Culture of Ovaries:
Rau (1956) attempted to culture pollinated ovaries of Phlox drummondii and studied the influence of extraneous chemicals on the pattern of development of the endosperm and embryo.
The addition of colchicine to the medium caused aberrant divisions, fusion and aggregation of endosperm nuclei and finally a degeneration of the endosperm. In 12 to 14-days old cultures the embryo also aborted and the seeds were malformed.
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Nirmala Maheshwari and Lal (1961) excised the flowers of Iberis amara one day after pollination and planted them on agar nutrient media. Fruits of normal size were obtained in two weeks on a medium containing Knop’s mineral salts + Nitsch’s trace elements + sucrose + B vitamins.
The seeds formed in vitro contained viable embryos. Removal of the sepals slowed down the growth of the ovaries showing that the calyx is by no means an unessential structure but has an important role in the physiology of the fruit.
Pollinated ovaries of a few other plants have also been reared into fruits, but when unpollinated ovaries are taken, the fruits are either not formed or are seedless.
Johri and Sehgal (1963) cultured the ovaries of Anethum graveolens at the zygote stage on White’s basal medium containing yeast extract and /or casein hydrolysate thirteen to twenty weeks later, a polyembryonate mass developed by the proliferation of the zygotic proembryo and ruptured the pericarp.
Further growth resulted in the production of multiple shoots some of which flowered in the test tube after about seven months. Thus this was possible to obtain several seedlings from a single fertilized ovule.
However, it is easier to maintain ovules in sterile culture when they are left in situ within the ovary. The physiological requirements of fertilized ovules do not appear to be species specific, since young ovules of widely different species have grown to mature seeds following transplantation on to the placenta of Capsicum fruits.
Culture of Ovules and Parts of Ovules:
White (1932) was the pioneer to culture the ovules. White and La Rue cultured ovules of Erythronium and Antirrhinum on White basic medium containing indole acetic acid lAA.
Nirmala Maheshwari and Lal (1961) cultured the ovules of Papaver somniferum excised six days after pollination when they contained only a 2-celled proembryo and a free nuclear endosperm. These grew to maturity in twenty days and even germinated and produced seedlings in the culture tubes.
Several genera, such as Citrus, Eugenia and Mangifera show the occurrence of nucellar embryos. Since they have the same genetical composition as the maternal parent, they are of much importance for the clonal propagation of desirable varieties.
Rangaswamy (1961) reported that if the nucellar tissue of Citrus microcarpa is grown on a suitable nutrient medium containing casein hydrolysate, it proliferates profusely and on subculturing produces embryo-like regenerants termed “pseudobulbils”, which can develop into seedlings so that an indefinite number of new plants can be obtained from a single nucellus.
A. N. Rao (1963) successfully germinated seeds of an interspecific hybrid of Vanda on a simple agar nutrient medium. Some of the fertilized ovules directly produced seedlings; others gave rise to a callus from which new plants arose subsequently.
Research Paper # 13. Culture of Endosperms:
It is somewhat more difficult to raise tissue cultures from the endosperm. However, Rangaswamy and Rao (1963) established a continuously growing tissue from the mature endosperm of Santalum album when they cultured it along with the embryo on White’s medium containing yeast extract, kinetin and 2, 4-D. On the other hand, the endosperm failed to callus when it was grown without the embryo.
Johri and Bhojwani (1965), also cultured the endosperm of Exocarpus cupressiformis on White’s medium containing indole actic acid lAA, kinetin and casein hydrolysate. If merely the endosperm devoid of the embryo was grown, it did not proliferate. In their experiments, the shoots were differentiated and not the roots.
