Haploids are defined as saprophytes with gametophytic chromosome number and have been produced in a variety of plant species using a variety of methods.Although, the significance of haploids in genetics and plant breeding has been recognized for long time, with the advent of new biotechnology it has received renewed emphasis, so that the production of haploids has become an important component of biotechnology programmes in different countries.
Although, haploids could be produced following delayed pollination, irradiation of pollen, temperature shocks, colchicine treatment and distant hybridization, the most important methods currently being utilized under biotechnology programmes include
(i) anther or pollen culture and ovule culture and
(ii) chromosome elimination following interspecific hybridization (bulbosum technique).
Factors Affecting Haploid Production
- A number of factors influence androgenesis in vitro. The genotype of the donor plant plays a significant role in determining the frequency of pollen plant production. Anther wall factors also support pollen embryo development.
Histological studies support this view. As induction of the pollen into embryoids occurs most easily within the confines of an anther, the anther wall seems to provide a nursing effect. There are two schools of thought regarding the role of the anther wall. One is that it may have a stimulatory effect on the growth of pollen embryos (probably due to the presence of enhanced levels of some amino acids such as glutamine and serine); the other view holds that it may emanate some inhibitory substances into the culture medium thereby blocking the growth of more pollen into embryos.
The culture medium also plays a vital role since the requirements vary with the genotype and probably the age of the anther as well as conditions under which donor plants are grown. The medium should contain the correct amount and proportion of inorganic nutrients to satisfy the nutritional as well as physiological needs of the many plant cells in culture.
In addition to basal salts and vitamins, hormones in the medium are critical factors for embryo or callus formation. Cytokinins (e.g. kinetin) are necessary for induction of pollen embryos in many species of Solanaceae except tobacco. Auxins, in particular 2,4-0, greatly promote the formation of pollen callus in cereals. For regeneration of plants from pollen calli, a cytokinin and lower concentration of auxin are often necessary.
Sucrose has been considered the most effective carbohydrate source which cannot be substituted by other disaccharides. Glucose can be used in anther culture in some cases but fructose is far less effective. The concentration of sucrose also plays an important role in induction of pollen plants. Activated charcoal is also added to the culture medium.
It helps in the removal of inhibitors from the agar used for gelling the medium. Another role assigned to activated charcoal is the adsorption of 5¬hydroxymethylfurfural, a product of sucrose dehydration during autoclaving, assumed to bean inhibitor of growth in anther cultures.
Certain organic supplements added to the culture medium often enhance the growth of anther cultures. Some of these include the hydrolyzed products of proteins such as casein (found in milk), nucleic acids, and others. Coconut milk obtained from tender coconuts is often added to tissue culture media. It contains a complex mixture of nucleic acids, sugars, growth hormones and some vitamins.
The physiological state of the parent plant plays a role in haploid production. Success in haploid induction is in part dependent on knowledge of the physiology of the pollen yielding plant. In various plant species it has been shown that the frequency of androgenesis is higher in anthers harvested at the beginning of the flowering period and declines with plant age.
This may be due to deterioration in the general condition of the plants, especially during seed set. The lower frequency of induction of haploids in anthers taken from older plants may also be associated with a decline in pollen viability. Seasonal variations, physical treatment, and application of hormones and salts to the plant also alter its physiological status, which is reflected in a change in anther response.
Temperature and light are two physical factors which play an important role in culture of anthers. Higher temperatures (30°C) yield better results. Temperature shocks also enhance the induction frequency of microspore androgenesis. Frequency of haploid formation and growth of plantlets are generally better in light.
Certain physical and chemical treatments given to flower, buds or anthers prior to culture, can be highly conducive to the development of pollen into plants. The most significant is cold treatment.
The developmental stage of pollen greatly influences the fate of the microspore, Androgenesis occurs when a microspore or pollen is induced to shift from a gametophytic pathway to a sporophytic pathway of embryo formation.
Anthers of some species (Datura, tobacco) give the best response if pollen is cultured at first. mitosis or later stages (postmitotic), whereas in most others (barley, wheat, rice) anthers are most productive when cultured at the uninucleate microspore stage (premitotic). Anthers at a very young stage (containing microspore mother cells m tetrads) or a late stage (containing binucleate, starch filled pollen) of development are generally ineffective, albeit some exceptions are known.
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