Culturing of plant organs on nutrient medium is called as organ culture.
It is of two types: 1. Vegetative Organs 2. Reproductive Organs.
1. Vegetative Organs (Growing from Vegetative Parts):
(i) Root Culture:
The culturing of root tips of aseptically germinated seeds in a nutrient medium is known as root culture (Fig. 15.3).
The root tip culture was developed by Robbins of USA and Kotte of Germany in 1922. Kotte cultured the root tips of maize and pea on a variety of nutrient media but he could not succeed in the root culture experiments. In 1934, White was successful in culturing the root tips of tomato plant. He prepared a medium in which the yeast extract was replaced by pyridoxine, thiamine and nicotinic acid of B-vitamin complex. This medium is commonly known as White’s medium. Later, this medium was proved as one of the best basic media for in vitro root culture experiments.
1. Root tip cultures increase our knowledge of carbohydrate metabolism, role of mineral ions, vitamins etc., in the growth of roots.
2. These cultures proved the dependence of roots on shoots for the growth hormones.
3. Root culture of Leguminous plants help us to study the process of nodule formation by nitrogen fixing bacterium i.e., Rhizobium sps.
4. Root cultures also help us in locating the biosynthesis of alkaloids by the roots and also to get increased production of such compounds ex- Hyoscyamine is an alkaloid commercially produced from the root cultures of Datura stramonium.
(ii) Leaf Culture:
This culture is obtained from excised young leaf primordia or immature leaves on a chemically defined medium. T.A Steeves and I.M. Sussex (1966) successfully cultured the leaf primodia of Ferns Ex- Osmunda. They have choosen the very young leaves or leaf primordial for the leaf culture. Mature leaves are not useful for the in vitro culture experiments (Fig. 15.4).
1. Leaf culture is useful to study the effects of various nutrients, growth regulators and environmental factors on leaf growth.
2. In Ferns, leaf culture helps us in studying the various developmental stages of sporangia.
(iii) Shoot Tip Culture:
The culturing of terminal portion of shoot tip with meristem is called shoot tip or meristem culture. Loo (1945) reported the culture of shoot tips Asparagus. Ball (1946) devised a method to identify the exact part of the shoot systems that gives rise to a whole plant. Morel and Martin (1952) showed that virus free plants can be obtained by this method. The excised stem tip or meristem can be cultured aseptically on nutrient medium. The meristem grows directly into a simple leafy shoot or multiple shoots (Fig. 15.5).
Importance of Shoot Tip Culture:
1. Generally the meristematic tissue is free from virus due to their fast mitotic activity. Therefore shoot tip culture of a plant is ideal for producing virus free stock.
2. Asexual or vegetative propagation of whole plants using shoot tip culture is known as micropropagation.
3. Many plants produce seeds that are highly heterozygous in nature. Such seeds are not suitable for storing genetic resources. Genetic resources of such plants can be stored by shoot tip culture.
4. Propagation of haploid plants. Haploid plants derived from anther/pollen culture always remain sterile until and unless they are made homozygous diploids. Meristem or shoot tip culture of haploid plants can be used for the propagation of such haploid plants.
2. Reproductive Parts (Growing from Reproductive Organs):
(i) Anther Culture:
The anther culture was first developed by Shimakura in 1934 while studying the physiology of meiosis. The first haploid callus was obtained from the pollen grains of Ginkgo biloba by Tulecke in 1953. Subsequently haploid tissues were initiated from a large number of gymnosperms and angiosperms. In 1964, Guha and Maheshwari from the University of Delhi reported the formation of direct embryos from the cultured anthers of Datura innoxia (Fig. 15.6).
The origin of these embryos was traced to pollen grains and the plants formed were haploids. Later, with the technique developed by Nitsch, it became possible to culture isolated microspores and generate haploids from them. It also became possible to double the chromosome number of microspores using chemicals like colchicine and generate homozygous diploids from cultures.
The technique of haploid cultures resulted in the selection of improved varieties of paddy, tobacco, wheat, tomato and mustard etc. Some excellent contributions in anther culture and pollen embryogenesis came from Sunderland and his co-workers. They developed float anther culture technique where the anthers with Uni or binucleate pollen were floated on a nutrient medium and after a period of time the anthers burst and released the pollen into the nutrient medium. These pollen grains later developed into embryoids.
One of the most significant developments in plant biology during last two decades has been the discovery of in vitro androgenesis i.e., the formation of sporophyte from the male gametophyte. e.g. Solanaceae and Gramineae members.
The haploid callus, embryo and plantlet may originate in any one of the following ways:
1. From the continued divisions of the vegetative cell of the pollen. The generative cell here plays no role and soon degenerates.
2. From the division and redivision of the generative cell, not the vegetative cell.
3. From the division products of both the vegetative and generative cells.
Importance of Anther and Pollen Culture:
1. Haploid plants derived from anther or pollen culture and useful in cytogenetic studies.
2. By comparing the heterozygous diploid with haploid homozygous diploid population recessive phenotypic characters can be identified very easily.
3. Critical genetic analysis of haploid population derived from individual microspores of pollen tetrad is very useful for the study of genetic recombination in higher plants.
4. The series of cell divisions and mode of differentiation (embryogenesis or organogenesis), starting from single cell (microspore) and ending in whole organism can be studied under microscope.
5. Double haploid, that are homozygous and fertile are readily obtained, ending the selection of desirable gene combination.
6. Culture of isolated pollen provides a novel experimental system for the study of factor controlling pollen embryogenesis of higher plants.
7. Study of meiotic behaviour of haploids provides valuable clues to measure chromosome duplication within a species and for understanding a phylogenetic relationship among species. It also provides information for the interpretation of chromosome homology.
8. Genetic analysis could be performed on haploid population to establish inheritance patterns.
9. The use of haploids in the production of monosomics, nullisomics and other aneuploids.
(ii) Ovary Culture:
Tissue culture raised from the ovaries of pollinated or unpollinated flowers is called ovary culture.
The technique of ovary culture was first developed by Nitsch (1951). He cultured the detached ovaries of Lycoperisicon esculentus, Cucumis anguria, Nicotiana tobacum and Phaseolus vulgaris on synthetic medium. The ovaries of Cucumis and Lycopersicon excised from pollinated flowers developed into matured fruits with viable seeds.
Maheshwari (1958) succeeded in rearing the ovaries of Iberis amara excised from flowers one day after pollination. Nitsch (1951) obtained seedless fruits of tomato from unpollinated ovaries cultured on a medium with synthetic auxins like 2, 4-D (2,4- dichlorophenoxy acetic acid) or NOA (2, Naphthoxy acetic acid) or 2,4,5-trichloro phenoxy acetic acid).
Sachar and Guha (1962) observed that the achenes of Ranunculus produced in the fields remain dormant for about a year, but the achenes formed in ovary culture germinated without any dormant period.
Technique:
1. Collect the pollinated or unpollinated flowers from a healthy plant.
2. Wash them thoroughly with tap water, dip into 5% Teepol solution for 10 minutes and again wash to remove the trace of teepol.
3. Transfer the flowers to laminar air flow cabinet, surface sterilize the flowers by immersing in 5% sodium hypochloride solutions for 5-7 minutes. Wash them with sterile distilled water.
4. Transfer the flowers to a sterile petridish using a flamed forceps and a surgical scalpel, dissect out the calyx, petals, anther filaments etc., leaving only the pistil. During isolation of pistils, care should be taken to ensure that the ovaries are not injured in any way. Damaged pistil should be discarded as they often form callus tissue from the damaged parts.
5. Place the ovaries on nutrient agar medium.
6. Incubate cultures at 25°C for 16 hrs in day light. The light is provided by fluorescent lamp.
1. To study the early development of embryo, fruit, different aspects of fruit physiology including respiration, maturation and disease.
2. To study the effect of phytohormones on parthenocarpic fruit development from the culture of unpollinated pistil.
3. To study the role of floral organs in the fruit development. It has been found that pollinated ovary produce the normal fruits in vitro, if the sepals are not removed before culture. If sepals are removed before culture, addition of sucrose (5%) is necessary to obtain satisfactory growth of ovaries in culture. In barley, lemma and palea are very important. In onion, the growth of ovaries without perianth is markedly inhibitory.
4. Ovary culture has been successfully employed to obtain hybrids of diploid Brassica chinensis and autotetraploid of B. pekinensis which are normally cross incompatible.
5. Ovary culture has also been successful in inducing polyembryony. Polyembryony may develop in culture from the various parts of the ovary. These poly embryos give rise to many shoots instead of a single plantlet.
6. The culture of ovaries of apomicts may, therefore, help in understanding the nature of stimulus provided by pollination.
(iii) Ovule Culture:
Ovule culture is an elegant experimental system by which ovules are aseptically separated from the ovary and grown on nutrient medium.
In 1932, White for the first time cultured ovules of Antirrhinum majus. However, the technique of ovule culture was perfected by Maheshwari in 1958 by culturing of Papaver somniferum. Maheshwari and Lai (1961) could raise ovule culture of Papaver somniferum from ovules excised 6 days after pollination. Seeds formed in these cultures germinated immediately.
1. Collect the open flower where the anthers are dehisced and pollination has taken place. To ensure the fertilization, collect the flower after 48 hours of anther dehiscence.
2. Separate sepals, petals, androecium etc., from the ovary containing either fertilized or unfertilized ovules.
3. Soak the ovary in 6% NaOCl solution.
4. Rinse the ovaries 3-4 times with sterile distilled water.
5. Ovules are gently prodded aseptically with the help of spoon shaped spatula by breaking the funicles at its junction with placental tissue.
6. The spatula with ovules is gently lowered into the sterile solid or liquid medium as the culture vial is slanted about 45°.
7. Incubate the ovule culture in either dark or light at 25°C.
Isolated ovule culture as early as the zygote of two to four celled pre-embryo stage is of considerable importance.
It has the following important applications:
1. Test Tube Pollination and Fertilization:
By this technique, it may be possible to germinate pollen in the same culture as the excised ovule and to induce in vitro fertilization. Excised unfertilized ovules of Argemone, mexicana, Papaver somniferum, Nicotiana tabacum have been cultured along with their respective pollen grains. All the stages of development starting from the germination of pollen to double fertilization have been observed and the mature seeds containing viable embryos have been obtained by this method. Using the same method, it has been possible to fertilize the ovules of Melandrium album with the pollen grains from other species of caryophyllaceae and subsequently even with pollen of Datura stramonium.
2. Application of Ovule Culture in Hybridization:
In many interspecific and intergeneric crosses, the F1-hybrid embryos frequently become abortive in the developing seeds or the F1 seeds are not capable to support the development of embryos. Ovule culture has been successfully employed to obtain hybrid seedlings in Abelmoschus and Gossypium etc.
3. Production of Haploid Callus through Ovule Culture:
Uchimiya et al. (1971) cultured unfertilized ovules of Solanum melangena and obtained vigorous callus formation on a medium supplemented with IAA or kinetin. Although the origin of the callus tissue was not known, a cytological observation revealed it to be haploid in nature. So, it is an important attempt to obtain a haploid cell line or plant from an alternative source rather than anther or pollen culture.
4. Ovule Culture and Angiospermic Parasites:
It is generally believed that in obligate root parasites such as Striga and Orobanche, the formation of seedlings is dependent on some stimulus from the host root. Studies on ovule culture of Orobanche have demonstrated that the formation of shoots in vitro can be induced in the absence of any stimulus from the host.
5. Ovule Culture in Orchid Plants:
In nature the seeds of orchids germinate only in association with a proper mycorrhizal fungus. Thus many seeds fail to give rise to plants. Further, the seeds of many orchids take long time to mature. To overcome such problems several attempts have been made to culture the fertilized ovule of orchids in vitro.
6. Induction of Polyembryony:
In horticultural practices, the artificial induction of polyembryony holds a great potential. The nucellus of monoembryonic ovule of citrus can be induced to form embryos in culture.
7. Virus Irradiation through Ovule Culture:
In citrus, the ovule culture has proved decisively advantageous to make them virus free.
(iv) 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. Harming (1904) for the first time obtained viable plants in vitro from excised embryos of Cochleria and Raphanus. Overbeek et al. (1941) discovered that coconut milk stimulated the growth of Datura embryos. This facilitated embryo culture in vitro. Randolf (1945) isolated Iris embryos to shorten the life cycle. In 1945 Cox and his co-workers grew embryos of different cultivars of cabbage to speed up seed germination.
In principle there are two types of embryo culture:
1. Culture of Immature Embryos Originating from Unripe Seeds:
This type of embryo culture is mainly used to avoid embryo abortion (early death of the embryo), with the purpose to produce a viable plant. This type of culture is difficult, not only because expertise in dissection is necessary, but also a complex nutrient medium is needed. The chances of success of this type of culture depend strongly on the developmental stage of embryo when it was isolated.
2. Culture of Mature Embryos Derived from Ripe Seeds:
This type of culture is relatively easy and is used to eliminate the inhibition of seed germination. The use of a simple nutrient medium with agar, sugar and minerals for this type of culture is sufficient.
If the development of immature embryos in vitro and in vivo is compared starting from the globular stage then the embryos in vitro regularly have:
i. A more bulky growth and are pear shaped.
ii. A longer globular stage.
iii. Initially only one cotyledon (in dicotyledonous plants) while two develop in vivo simultaneously.
iv. The possibility of showing polycotyledons development (more than two cotyledons).
In vitro grown embryos usually exhibit early germination (Jensen 1976) since there is a loss of inhibitors when the seed coat is removed, another reason might be that the (negative) osmotic potential in vivo has a much higher value than in vitro.
Techniques:
With embryo culture there are normally no problems with disinfection. Single mature seeds are externally disinfected, then embryo is removed after the seed coat is cut open. The dissection of the embryos produces more problems. It is possible to dissect large embryos without the use of a microscope, but this must be used with a cold light source, for small embryos.
Inoculation needles and empty holders with a piece of razor blade mounted at the end are used during the dissection procedures. Care should be taken when cutting the seed as it is easy to see where the small embryos are located and consequently there is a less chance of damaging them, although care should be taken not to damage the suspensor.
Isolation of cherry embryos is as follows:
First of all the stones are removed from the fruits, then the stones are placed into a water bath to see which embryos are good (those stones that sink in water). These are then disinfected. The sterile cherry stones are then cracked and the embryo can be seen. The embryos are then removed with the help of pointed forceps and inoculated on to a solid medium.
Application of Embryo Culture:
1. Due to the inhibition of seed germination in a few species it is absolutely impossible to obtain germination in vivo. In these cases embryo culture is essential. Musa balbisiana Pinus armandii × P.Koraiensis and Colocasia esculenta.
2. Seeds of obligate parasites will not be able to germinate without host, but it can be achieved with embryo culture. Johri and Bajaj (1962) successfully cultured the young embryo of Cuscuta reflexa.
3. Shortening of the breeding cycle or life cycle. The life cycle of Iris was reduced from 2-3 years to less than one year by using embryo culture techniques.
4. Production of Haploids- With the cross Hordetim Vulgare × H.bulbosum, fertilization occurs but there after the chromosomes of H. bulbosum are eliminated. The result is that the haploid embryo of H.Vulgare remains which is viable only by embryo culture.
5. Prevention of embryo abortion with early ripening of stone-fruits like cherry, peach, apricot and plum. The transport of water and nutrients to the yet immature embryo is sometimes cut off too soon, resulting in the abortion of the embryo. To overcome this problem embryo culture is the only solution.
6. The prevention of embryo abortion as a result of incompatibility with interspecific crosses, intergeneric crosses and with crosses between diploids and tetraploids the endosperm often develops poorly or not at all. The results in embryo abortion in vivo, which can only be avoided by embryo culture.
7. Vegetative propagation- The embryos of gramineae and conifereae are often used as a starting material for vegetative propagation.
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