Haploids are plants with a gametophytic chromosome number and doubled haploids are haploids that have undergone chromosome duplication. The production of haploids and doubled haploids (DHs) through gametic embryogenesis allows a single-step development of complete homozygous lines from heterozygous parents, shortening the time required to produce homozygous plants in comparison with the conventional breeding methods that employ several generations of selfing. The production of haploids and DHs provides a particularly attractive biotechnological tool, and the development of haploidy technology and protocols to produce homozygous plants has had a significant impact on agricultural systems.
There are several available methods to obtain haploids and DHs, of which in vitro anther or isolated microspore culture are the most effective and widely used.
Haploid plants are sporophytes carrying the gametic chromosome number (n instead of 2n).
Anther culture is a technique by which the developing anthers at a precise and critical stage are excised aseptically from unopened flower bud and are cultured on a nutrient medium where the microspores within the cultured anther develop into callus tissue or embryoids that give rise to haploid plantlets either through organogenesis or embryogenesis.
The basic principle of anther and pollen culture is the production of haploid plants exploiting the totipotency of micro spore and the occurrence of single set of chromosome (n) in micro-spore. In this process, the normal development and function of the pollen cell to become a male gamete is stopped and is diverted forcibly to a new metabolic pathway for vegetative cell division.
- For this objective, microspores, either within intact anther or in isolated state, are grown aseptically on the nutrient medium where the developing pollen grain will form the callus tissue or embryoids that ultimately give rise to haploid plantlets.
- In fact, anther culture is in essence the pollen culture. The principle behind the anther culture is that without disturbing the natural habitat and environment of the enclosed anther, pollen can be grown by culturing the intact anther. In culture condition, the diploid tissue of anther will remain living without proliferation at the selective medium and, at the same time, it will encourage the development of pollen by nursing and providing nutrient.
The haploid embryoids or the callus tissue can be seen as the anther dehisces in culture. But there is always the possibility that the diploid somatic cells of the anther will also respond to culture condition and so produce unwanted diploid callus or plantlets.
- In attempts to avoid this problem, free pollens isolated from the anther are grown in nutrient medium. The knowledge gained so far from anther and pollen culture has established that pollens at the uni-nucleate stage, just before the first mitosis, or during mitosis are most suitable for the induction of haploids.
Induction of haploids can be enhanced by keeping the anther or flower bud at low temperature. The low temperature has been ascribed to a number of factors such as dissolution of microtubules, alteration in the first mitosis or maintenance of higher ratio of viable pollen capable of embryogenesis. Cold treatment may also act to help the embryogenesis by repressing the gametophytic differentiation or by lowering the abscisic acid content of the anther which is considered to be inhibitory for the production of haploids.
The important aspect of anther- or pollen culture is the nutrient medium. The nutritional requirements of the excised anther are much simpler than those of isolated microspores. In the isolated microspore, it is obvious that certain factors responsible for the induction of haploids, which might have been provided by the anther, are missing and these have to be provided through the medium.
In culture, pollen may divide mitotically or can follow the normal pathway of forming vegetative and generative nuclei. The generative nucleus remains quiescent and abort. The vegetative nucleus divides repeatedly, forming a multinucleate pollen. The multinucleate pollen undergoes segmentation which may lead to form either organised embryoid structure or callus tissue. Both types of development are utilised to form haploid plantlets.
The haploid plantlets are self-sterile due to presence of single set of chromosome which are not able to participate in meiotic segregation. By colchicine treatment, haploids are made homozygous diploid, or isogeneic diploid which are fertile. Haploids or homozygous diploid grown in vitro are transferred to pot and grown to maturity in the glasshouse.