3. Gynogenesis
Gynogenic development of plants from unfertilized cells of female gametophyte (embryo-sac) in ovary/ovule/young flower cultures is one of the available alternatives for haploid production. It was first reported in barley San Noeum (1976). This method of haploid production is more tedious than androgenesis. The reasons for this being the indefinite numbers of microspores (male gametes) within the anther wall for androgenesis as against single egg cell (female gamete) per flower for gynogenic haploid production, which too, is deep seated within the embryo-sac (female gametophyte), thus making the entire process very cumbersome. The technique is very useful where anther culture has been unsuccessful, plants are male sterile or androgenesis is confronted with the problem of albino or non-haploid formation. The following techniques are generally used for production of haploids via in vitro gynogenesis either through direct embryogenesis or via callusing.
3.1. In situ parthenogenesis induced by irradiated pollen followed by in vitro embryo culture
Parthenogenesis induced in vivo by irradiated pollen, followed by in vitro culture of embryos can be an alternative method of obtaining haploids in fruit crops. Gynogenesis by in situ pollination with irradiated pollen has been successfully used for Malus domestica (L.) Borkh, Pyrus communis L., Actinidia deliciosa (A. Chev). This method is based on in vitro culture of immature seeds or embryos, obtained as a result of pollination by irradiated pollen with gamma rays from cobalt 60. The method is useful in those species in which in vitro anther culture has not been successful. Irradiation does not hinder pollen germination but prevents pollen fertilization and, thereby, stimulating the development of haploid embryos from ovules. The success of this technique is dependent on the choice of radiation dose, the developmental stage of the embryos at the time of culture, culture conditions and media requirements.
3.2. Ovary slice culture
Ovary slice culture technique involves culture of transverse sections of unpollinated ovaries on culture media. The following protocol was used to induce in vitro gynogenesis in Tea (Hazarika and Chaturvedi 2012):
- a. For Ovary slice culture in Tea, unopened and unpollinated mature flower-buds (6-10 mm) size were collected early in the morning. Some of the buds were fixed in FAA (5:5:90 v/v/v Formaldehyde: Acetic acid: 70% Ethanol), for 48 h, and then stored in 70% alcohol. Later on, the appropriate developmental stage of the embryo sac was determined by histological analysis.
b. The flower buds were surface sterilized with 0.1% HgCl2 for 7 minutes, followed by rinsing with sterile distilled water at least thrice.
c. Carefully dissected transverse sections of ovaries were cultured on Murashige and Skoog's media supplemented with varying concentrations of Auxins and Cytokinins.
d. Six ovary slices containing unpollinated ovules were cultured in 60 mm X 15 mm pre sterilized disposable Petriplates containing 10 ml MS medium.
e. The sealed Petriplates were subjected to various regimes of temperature and light treatments.
3.3. Ovule culture
The unfertilized ovary is surface sterilized and the ovules were taken and placed into culture. Excision of ovule, followed by culture on specific media may be either extremely easy to accomplish, as in case of large-seeded species in which only a single ovule is present, or time-consuming and intricate, in small-seeded polyovulate species. Two types of ovule support systems have been developed. The filter paper support system involves culturing of the ovules on top of filter paper placed over liquid medium, whereas the vermiculite support technique demands placing the ovules on a sterile vermiculite/liquid media mixture (vermiculite support) with the micropylar side down. Unpollinated ovule culture has been used for haploid production in sugar beets and onions.