Anther Culture:

It is a relatively simple and efficient method, requiring minimum facilities. Flower buds, with pollen grains at the most labile stage are surface sterilized and the anthers, excised under aseptic conditions, cultured on semi-solid or in liquid medium. In some cases, where the flower buds are small, whole buds or inflorescences enclosing the anthers at the appropriate stage of pollen development are cultured. The cultures are exposed to pretreatments, such as low or high temperature shock, osmotic stress or nutrient starvation, before incubation at 25℃ in the dark. Depending on the plant species, and to some extent the culture medium, the androgenic pollen either develops directly into embryos or proliferates to form callus tissue. After 2–3 weeks, when pollen embryos become visible, the cultures are transferred to light for their further development and organogenic/embryogenic differentiation. The shoots regenerated from callus often require transfer to another medium for rooting to form complete plants.

Pollen/Microspore Culture:

It is now possible to achieve androgenesis in cultures of mechanically isolated pollen of several plants, including tobacco, Brassica species and some cereals. In addition to the culture medium and pretreatment, the plating density (number of pollen grains per unit volume of medium) is a critical factor for the induction of androgenesis in cultured pollen. Treatment of pollen-derived embryos and pollen-derived callus to recover complete plants is the same as in anther culture. The nutritional requirements of isolated pollen in culture are more complex than those of cultured anthers. However, unlike the earlier belief, pollen culture is less tedious and time consuming than anther culture. The additional advantages of pollen culture over anther culture for haploid plant production are as follows:

·       Homogeneous preparation of pollen at developmental stage is most suitable for androgenesis which can be obtained by gradient centrifugation.

·       Isolated pollen can be modified genetically by mutagenesis or genetic engineering before culture, and a new genotype can be selected at an early stage of development.

·       Pollen culture improves considerably the efficiency of androgenesis. In rapid cycling Brassica napus, the culture of isolated pollen was 60 times more efficient than anther culture in terms of embryo production.

·       The exogenous treatments can be applied more effectively and their precise role in androgenesis studied as the unknown effect of the anther wall is eliminated.

·       The culture of isolated pollen provides an excellent system to study cellular and subcellular changes underlying the switch from gametophytic to sporophytic development and the induction of embryogenesis in isolated haploid single cells.

  Factors affecting in vitro androgenesis:

Some of the factors that have a profound effect on the fate of pollen in culture are the genotype and the physiological state of the donor plants, the developmental stage of pollen at the time of culture, pretreatments, and the culture medium. Before giving general protocols for anther and pollen culture the effects of these factors on androgenesis are described to facilitate modification of the available protocols to optimize the androgenic response of any specific system.

 Genotype: The androgenic response is influenced considerably by the plant genotype. The observed interspecific and intraspecific variation is often so great that while some lines of a species are highly responsive, others are extremely poor performers or completely non-responsive. Since plant regeneration from pollen is a heritable trait, it is possible to improve the androgenic response of poor performers by crossing them with highly androgenic genotypes. In general, indica cultivars exhibit poorer response as compared to japonica cultivars of rice.

 Physiological status of the donor plants: The environmental conditions and the age of the plant, which affect the physiology of the plants, also affect their androgenic response. Generally, the first flush of flowers yields more responsive anthers than those borne later. However, in B. napus and B. rapa, pollen from older, sickly looking plants yielded a greater number of embryos than those from young and healthy plants. Similarly, the late sown plants of B. juncea yielded more androgenic anthers than the plants sown at the normal time.

Pretreatments: Application of a variety of stresses, such as temperature shock, osmotic stress and sugar starvation at the initial stage of anther or pollen culture have proved promotory or essential for the induction of androgenesis. However, the type, duration and the time of application of these pretreatments may vary with the species or even the variety. Ex- Initial starvation of developing pollen of important nutrients, such as sucrose and glutamine favoured androgenesis in tobacco and barley.

Stage of pollen development: The competence of pollen to respond to the various external treatments depends on the stage of their development at the time of culture. Generally, the labile stage of pollen for androgenesis is just before, at, and immediately after, the first pollen mitosis. During this phase, the fate of the pollen is uncommitted because the cytoplasm is cleaned of the sporophyte-specific information during meiosis, and the gametophyte-specific information has not been transcribed by this time. However, it is important to appreciate that the most vulnerable stage of pollen for responding to exogenous treatments may vary with the system.

 Culture medium: The most widely used media for this purpose are MS (Murashige and Skoog 1962), N&N (Nitsch and Nitsch 1969) and their modifications. In general, the requirements of isolated pollen cultures are more elaborate than that of anther cultures. Anther cultures of many cereals are very sensitive to inorganic nitrogen, particularly in the form of NH4⁺. Indica rice anther and pollen cultures are even more sensitive to the concentration of NH4 + in the culture medium. Sucrose is an essential constituent of media for androgenesis and it is used mostly at 2–4% (w/v).

 Applications of Androgenesis

·       Haploids and DHs are of considerable importance in genetics and plant breeding programs. The main advantage of haploids in plant breeding programmes is to achieve complete homozygosity in a single step by doubling the chromosome number. Combining the conventional breeding methods and anther culture, Chinese scientists have developed and released several cultivars of rice (38 varieties), wheat (10 varieties), and maize (22 varieties), which have good agronomic characteristics, such as high yield, well adaptation, and resistance.

·       Haploids with desirable mutations can be selected and their chromosomes duplicated by colchicine treatment to get fertile diploids with all desirable mutations in a single generation.

·       Androgenesis is a good source of genetic variation or gametoclonal variation. It enables analysis of gametic variation, generated through recombination, and segregation during meiosis, at plant level.

·       Androgenesis provides an excellent model system to study the developmental aspects of induction of embryogenesis and embryo development from single haploid microspores.

·       The doubled haploids of self-pollinating crops (barley, tomato, wheat) have been used to construct linkage maps of molecular markers and to estimate the number of genes controlling complex traits and even to directly release cultivars. In outbreeding crops (maize, potato, cucumber), DHs have been applied to study the effect of inbreeding and to derive inbred lines. Such lines can be used for hybrid cultivar production or for studying the inheritance of useful  traits.

 Conclusion:

Androgenic haploids have been produced for a very large number of angiosperms, and the technique of anther/pollen culture for haploid production has become an integral part of plant breeding programs. Moreover, androgenesis is not applicable to male sterile or female clones of the dioecious plants. The main factors that hinder the application of anther and pollen culture to cereals are low rates of androgenesis and the high frequency of albinism (up to 80 %) among the regenerants. The albino plants cannot survive in the field, and therefore are of no agronomic value. Nuclear fusion and endomitosis during early stages of androgenesis, especially in cereals, results in non-haploids. These are some of the limitations of in vitro androgenesis that need to be addressed by the scientists.

Refrences

Bhojwani SS, Dantu PK (2010) Haploid plants. In: Davey MR, Anthony P (eds) Plant cell culture: essential methods. Wiley-Blackwell, UK

Bhojwani SS, Razdan MK (1996) Plant tissue culture: theory and practice, a revised edition. Elsevier, Amsterdam

Blakeslee AF, Belling J, Farnham ME, Bergner AD (1922) a haploid mutant in the Jimson weed Datura stramonium. Science 55:646–647

Guha S, Maheshwari SC (1964) In vitro production of embryos from anthers of Datura. Nature 204:497.