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4.4. Anther wall factor
One of the important research subject in anther culture of woody plants is to avoid the over proliferation of callus from anther wall tissues and to achieve a high yield of pollen embryoids and pollen calli. In anther culture of most woody plants, both pollen calli (or embryoids) and somatic calli from anther wall tissues grew simultaneously. The development of callus from somatic tissues of anther can be avoided by culture of isolated microspores. However, microspore culture is not very successful in woody plants (Chaturvedi et al, 2003). Pelletier and Ilami (1972) had introduced the concept of “Wall Factor”, according to which the somatic tissues of anther play an important role in the induction of sporophytic divisions in pollen. In anther culture of rubber, 47% of the microspores in close contact with the surrounding somatic cells could develop into multicellular masses as compared to only 5% of microspores away from the wall (Chen 1986). Anther wall callusing was regarded as a pre-requisite for the formation of androgenic haploids (Chaturvedi et al, 2003; Chen 1986; Chen et al, 1982 ).
4.5. Chemical factors
The constituents of the basal medium and combination/s of growth regulators serve as important factors in eliciting successful androgenesis or gynogenesis. However, it is difficult to suggest one culture medium or one growth regulator for all the systems. The requirement of growth regulators and culture medium in terms of kind and concentration may vary with the system. Generally, there is an agreement that the source and amount of total nitrogen as well as a combination of a cytokinin and an auxin is necessary for pollen embryogenesis and pollen callusing in several woody plants (Chaturvedi et al, 2003; Chen 1986; Nair et al, 1983). Sucrose has generally been used as the major carbohydrate source in the culture medium. The effect of sucrose on anther culture has been investigated in a number of species. Generally, sucrose is supplied at 2-3% concentration. However, increase in its concentration can lead to beneficial morphogenic potential by suppressing the proliferation of somatic tissues.
4.6. Effect of female flower position
Position of female flowers on the plant stem affected induction of embryos from ovule cultures of Cucurbita pepo L (Shalaby, 2007). One of the possible explanations for enhancing responses of tissue culture could be attributed to indigenous hormonal level (Johansson, 1986).
5. Diploidization of haploids
The haploids may grow normally up to flowering stage but in the absence of homologous chromosomes the meiosis is abnormal and consequently, viable gametes are not formed. To obtain fertile homozygous diploids, the chromosome complement of the haploids must be duplicated. For long colchicine has been used for the purpose and is more effective.
Another method to diploidise the haploids is by utilizing the tendency of in vitro growing callus cells to undergo endomitosis. Segments from vegetative parts of haploid plants are grown on a suitable culture medium and made to proliferate into calli. After some time many of the callus cells become diploid due to endomitosis. By transferring such calli to an appropriate medium fertile diploids shoot can be obtained.