4. Factors affecting haploid androgenesis
There are numerous endogeneous and exogeneous factors that affect in vitro haploid production. These factors can be genetic, physiological, physical and chemical may also interact amongst each other to divert the microspores/egg cell to enter into a new developmental pathway. Some of the crucial factors affecting haploid production in plants have been discussed below:
4.1 Genotype of the donor plant
The genotype of the donor plants, i.e. genetic factor, has a great influence on the anther, ovary and ovule culture response. In earlier studies, significant difference in callus formation using varieties or crosses were observed. In some species only a few genotypes have responded of many tested. In fact genetic factors contribute in a major way to the differences in the number of haploid plants produced (Custódio et al, 2005).
4.2 Physiological status of the donor plant
The physiological conditions of the donor plant, i.e the environmental conditions and age of the donor plant, directly affects both in vitro androgenesis and in vitro gynogenesis in almost all plant species. A correlation between plant age and anther response has also been demonstrated. Similar is the case with ovary culture. The frequency of androgenesis is usually higher in anthers harvested at the beginning of the flowering period and showed a gradual decline in relation to plant age (Bhojwani and Razdan 1996). Varying temperature and light conditions during the growth of donor plants also affect anther response. In anther culture of grape, the induction frequency of embryoids derived from spring flowers was higher than that derived from summer flowers (Zhou and Li 1981). The microscopical observations showed that some varieties of rubber tree often have a lot of degenerated and sterile microspores in their anthers in early spring or hot summer due to the influence of unfavourable climatic conditions (Chen et al, 1982). As a result no pollen embryoids were obtained from such anthers but only the somatic calli.
4.3 Stage of explants material at the time of inoculation
4.3.1. Stage of microspores
The stage of microspores at the time of inoculation is one of the most critical factors in the induction of androgenesis. Detailed cytological studies conducted on poplars, rubber (Chen 1986) and apple (Zhang et al, 1990) have shown that androgenic callus and embryos were mainly induced through a deviation of the first pollen mitosis to produce two undifferentiated nuclei. Besides affecting the overall response, the microspore stage at culture also has a direct bearing on the nature of plants produced in anther culture (Sunderland and Dunwell 1977). About 80% of the embryos obtained from binucleate microspores of Datura innoxia, a highly androgenic species, were non-haploids (Sunderland et al, 1974). In a vast majority of species where success has been achieved, anthers were cultured when microspores were at the uninucleate stage of microsporogenesis (Chaturvedi et al, 2003; Pedroso and Pais 1994; Sopory and Munshi 1996).
4.3.2. Stage of the embryo sac
It has been reported that the effect of ovule development on gynogenesis is profound as it harbours the embryo sac comprising of the egg cell. The stage of embryo sac is an important determining factor for in vitro gynogenesis in various plant species. However, it is difficult to know the stage of embryo sac at the time of inoculation. Several authors prefer to describe the inoculation stage according to the developmental stage of the flower bud or stage of pollen development. However, this could not be possible in several species, where male and female gametophytes do not mature simultaneously, a phenomenon known as protandry, the maturation of anthers before carpels (e.g., onion, leek, sunflower, sugar beet and carrot) and the opposite protogyny (e.g., pearl millet). In such cases, the stage of embryo-sac at culture can be determined by histological preparations of ovary/ovules that are at identical stage with that of cultured ovary/ovules.
Although a wide range of embryo sac stages are responsive to gynogenic development, but, in most cases nearly mature embryo-sac stage gave better results. This is quite contrary to anther culture in which mature pollen is non responsive to androgenesis. In Barley and rice, unfertilized ovary cultures with late staged mature embryo sacs gave good results (San Noeum 1976, 1979; Wang and Kuang 1981) while others reported success with ovary cultures containing uninucleate to mature embryo sacs (Zhou and Yang, 1981b, 1982; Kuo, 1982; Huang et al., 1982).