Types of culture
It deals with the culture of the isolated organs (roots) under laboratory conditions (in vitro)
. Different names are given depending upon the organ used for the culture. For instance the culture of roots, endosperm, ovary, and ovule are called as root culture, endosperm culture, ovary culture etc. It was Skoog (1944), who for the first time suggested that the organogenesis could be chemically controlled. Skoog and Miller (1957) also demonstrated that a high ratio of auxin: cytokinin stimulated the formation of root in tobacco callus, but a low ratio of the same induced shoot information.
The culture of plant parts (explants) is known as explant culture. The explants can be any part of the plant e.g. the piece of stem, leaf, hypocotyl, etc. The explant cultures are generally used to induce callus or plant regeneration.
Callus refers to an unorganized mass of cells generally parenchymatous in nature.
The unique feature of callus is that the abnormal growth has biological potential to develop normal root, shoots, and embryoids, ultimately forming a plant. Naturally, the callus is formed due to the infection of microorganisms from wounds due to stimulation by endogenous growth hormones, the auxins and cytokinins. However, it has been possible to artificially develop callus by using tissue culture techniques.
Auxins are added to culture medium for callus induction but the nature and quantity of auxin added, depends on the nature and source of explant and its genotype besides other factors. Callus cultures can be maintained for prolonged periods by repeated sub-culturing. Callus cultures are used for a) plant regeneration, b) preparation of single cell suspensions and protoplasts, and, c) genetic transformation studies.
Factors affecting Callus culture
- the source and the genotype of the explant
- composition of the medium (most commonly used-MS medium)
- temperature (22-280C suitable for callus formation)
- growth regulators e.g. auxins, cytokinins alone or combination of these.
- Age of the plant
- Location of the explant
- Physiology and growth condition of the plant
Cell suspension cultures
Cell suspension is prepared by transferring a fragment of callus to the liquid medium and agitating them aseptically to make the cells free.
Single cells can be isolated from either callus or any other part of the plant and cultured in liquid medium using both mechanical and enzymatic methods. Mechanical methods involve grinding of the tissue to a fine suspension in a buffered medium followed by filtration and centrifugation to get rid of cell debris. The enzymatic method uses the enzymes (pectinase or macerozyme) to dissolve the middle lammela between the cells. After the isolation of the cells, they are cultured by batch cultures or continuous cultures.As the medium is liquid in nature, the pieces of callus remain submerged which creates anaerobic conditions. To overcome this problem, the suspension cultures are agitated by a rotary shaker which disperses the cells and expose them to air.
The advantages of cell suspension cultures over the callus culture:
a) The suspension can be pipetted.
b) They are less heterogeneous and cell differentiation is less pronounced.
c) They can be cultured in volumes upto 1,500 litres.
d) They can be subjected to more stringent environmental controls.
e) The manipulations for the production of natural products by feeding precursors, is possible.
Batch cultures are initiated as single cells in 100- 250 ml flasks and are propagated by transferring regularly small aliquots of suspension to a fresh medium. Continuous cultures are maintained in a steady state for long period by draining out the used medium and adding fresh medium.
The cell suspension cultures can be used for a) induction of somatic embryos/shoots, b) in vitro mutagenesis and mutant selection, c) genetic transformation, d) production of secondary metabolites.
Mass cell culture
Plant cells are cultured in specially designed 'plant bioreactors' which essentially do not have a stirrer as plant cells are shear sensitive. In place of stirrer, gas is gently bubbled which provides stirring as well as meet the demand of a higher oxygen supply.
Protoplasts are plant cells without cell wall and can be isolated by using enzymes like cellulases, pectinases) from leaf, seedling, calli, pollen grains, embryo sacs etc.The protoplasts regenerate cell wall, undergo cell division, and form callus. The callus can also be subcultured. Some of the examples of plant species that have been regenerated from protoplasts are--- Cucumis sativus, Capsicum annum, Ipomoea batata, Glycine max, Chrysanthemum sp. These cultures are used for a) various biochemical and metabolic studies, b) fusion of two somatic cells to create somatic hybrids, c) fusion of enucleated and nucleated protoplasts to create Cybrids (cytoplasmic hybrids) and d) genetic manipulation. e) drug sensitivity.
Bergmann’s cell plating technique (culture of single cells)
In this technique, free cells are suspended in a liquid medium. Equal volumes of liquid and agar media are mixed and rapidly spread in petri dish, which makes the cells evenly distributed in a thin layer after solidification. After sealing the Petri dishes with parafilm, they are examined under the inverted microscope to mark the single cells. Plates are incubated in dark at 250C and cell colonies developing from marked single cells, are used to obtain single cell cultures.
Besides, roots, shoots, and pollen, embryos can also be cultured to produce haploid plants. The embryo culture is very useful in conditions where embryo fails to develop due to degeneration of embryonic tissues. It has been used as a routine technique in orchid propagation, in breeding of species showing dormancy.
It has been observed that sometimes, inspite of successful pollination and fertilization, the embryos do not develop. The incompatibility between the embryo and the endosperm or some inherent deficiency also results in the under development of embryo. These immature embryos can be dissected out from the seeds and can be grown artificially on culture medium. These embryos differentiate into shoot, root and plantlets under culture conditions. This technique of growing immature embryo is termed as ‘embryo rescue’. This technique is very useful in hybridization, breaking dormancy of certain seeds, and to achieve complete growth of embryo into a plant.
Anther and Pollen Culture (Production of haploid plants)
Haploid plants possess a single set of chromosomes (gametophytic no of chromosomes i.e. n) in the sporophyte in contrast to diploids which contain two sets of chromosomes (2n). The existence of haploid plants was reported by Bergner (1921) in Datura stramonium. Tulecke (1951) cultured the pollen grains of Ginko biloba (gymnosperm) and succeeded in inducing the development of haploid callus. Guha and Maheswari (1964) reported the direct development of haploid embryos and plantlets from microspores of Datura inoxia by the cultures of excised anthers. In 1967, Bourgin and Hitsch obtained the first full haploid plants from Nicotiana tabacum.
Haploid plants are very useful in:
a) direct screening of recessive mutation because in diploid or polyploid screening of recessive mutation is impossible.
b) Development of homozygous lines in a short span of time.
c) The generation of exclusive male plants by the process of androgenesis using techniques to double the chromosome numbers.
d) Production of disease resistant plants by introducing disease resistant genes e.g. Barley accession Q 21681 resistant to stem rust, leaf rust, and powdery mildew.
e) Cytogenetic research which includes production of aneuploids, determination of origin and basic number of chromosome numbers, induction of genetic variability.
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 e.g. rice, wheat, maize, coconut, rubber trees etc. The Institute of Crop Breeding and Cultivation (China) has developed the high yielding and blast resistant varieties of rice zhonghua No.8 and zhonghua No. 9 through transfer of desired alien gene.
In androgenesis, the male gametophyte (the microspore or immature pollen) produces haploid plants by stopping the development of pollen cell into a gamete and forcing it to develop into a haploid plant.
In vitro Androgenesis
In vitro androgenesis is the formation of sporophyte from the male gametophyte on artificial medium and is most commonly found in family Solanaceae and Poaceae (Graminae).
In the pollen derived embryogenesis, also called direct androgenesis, the pollen directly acts as a zygote and passes through various embryogenic stages similar to zygotic embryogenesis. Direct androgenesis is very common in many plants of the family Solanaceae and Brassicaceae.
The process where the pollen grains instead of normal embryogenesis, divide erratically to develop callus is called indirect androgenesis e.g. in barley, wheat, Coffee etc.
The technique of anther culture
The anthers with their filaments are removed from the flower buds after surface sterilization. Under aseptic conditions, the anthers are excised and crushed in 1% acetocarmine to test the stage of pollen development. The anthers in correct stage of development are separated and inoculated on a nutrient medium. The anther cultures are maintained at 280C and alternating photo periods of light (12-18 hrs) and darkness (6-12 hrs). The anthers proliferate and produce callus which forms an embryo and the embryo subsequently develops into a haploid plant.
The Technique of Pollen Culture
The pollens are extracted by pressing and squeezing the anthers with a glass rod against the sides of the beaker. The anther tissue debris is removed by filtering the pollen suspension and large and healthy pollen are washed and collected. These pollen are cultured on a solid or liquid medium and the callus or the embryo formed is transferred to a suitable medium to produce a haploid plant.
The factors that affect androgenesis are the genotype and the physiological state of the donar plants, composition of the culture medium, the method adopted in the pretreatment of anthers to develop in to haploid plants and the stage of the microspore or pollen selected for culture.
The haploid are identified by looking at their morphological features or by using genetic markers e.g. a1marker for brown coloured aleurone to detect haploids in maize plant.
Limitations in Haploid production
a) Due to the low frequency of haploid production the selection is very difficult.
b) Haploids with deleterious traits frequently develop in cultures.
c) It is sometimes difficult to isolate haploids from the culture since the polyploids outgrow haploids.
d) The doubling of haploids (diploidization) does not always lead to the formation of homozygous plant.
e) The embryos derived from haploids often get aborted.