Elementary plant Biochemistry and Biotechnology (2+1)

Pollen or Microspore culture

 
• The pollen grains are released from the cultured anthers either mechanically. Or the cold treated anthers cultured on liquid medium burst open after 2-7 days liberating the pollen grains into the medium. This is called ‘float culture method’ which has proved better than mechanical isolation of pollen from fresh or pre-cultured anthers.


• To improve the efficiency of isolated pollen culture for the production of haploids, Wenzel and his colleagues introduced the technique of density gradient centrifugation which allows the separation of embryogenic grains from a mixture of embryogenic and non-embryogenic grains obtained after crushing the anthers. The anthers of Barley obtained at the proper stage of development and gently macerated to obtain a suspension of pollen grains.


• After removing the debris by repeated filtration and centrifugation, the suspension was layered on 30% sucrose solution and centrifuged at 1200 g for 5 min. The androgenic, vacuolated pollen grains formed a band at the top of the sucrose solution. Isolated pollen culture is not only more efficient but also more convenient than anther culture. The tedious process of dissection of anthers is avoided. Instead, the entire buds within a suitable size range are crushed and the embryogenic grains are then separated by gradient centrifugation (Figure).


Pathways of development

• The early divisions in responding pollen grains may occur in one of the following four ways (Figure).
• Pathway I: The uninucleate pollen grain may divide symmetrically to yield two equal daughter cells both of which undergo further divisions. (Dature innotura)
• Pathway II: In some other cases (Nicotiana tabacum, Datura metel, Triticale), the uninucleate pollen divides unequally (as it does in nature). The generative cell degenerates immediately or after undergoing one or two divisions. The callus/embryo originate due to successive divisions of the vegetative cells.
• Pathway III: But in some species like Hyoscyamus niger, the pollen embryos originate from the generative cell alone; the vegetative cell either does not divide or divides only to a limited extent forming a suspensor like structure.
• Pathway IV: In certain species such as Datura innoxia the uninucleate pollen grains divide unequally, producing generative and vegetative cells, but both these cells divide repeatedly to contribute to the developing embryo/callus.


• Pollen grains of many crop species, e.g. Tobacco, Wheat, Barley etc., exhibit pollen dimorphism. Most of the pollen grains are bigger, stain deeply with acetocarmine and contain plenty of starch. But small portions (ca. 0.7%) of the pollen grains are smaller and stain faintly with acetocarmine; these are called S-grains. These S-grains only respond during anther culture. The frequency of responding pollen grains can be enhanced over that of S-grains by certain pretreatments. E.g. chilling. Pollen grains of the cultured anthers show remarkable cytological changes during the first 6-12 days, called the inductive period. In tobacco, the gametophytic cytoplasm of binucleate pollen grains is degraded, ribosomes are eliminated and only few mitochondria and plastids remain. New ribosomes are synthesized following the first sporophytic division of the vegetative cell.


• The responsive pollen grains become multicellular and ultimately burst open to release the cell mass. This cell mass may either assume the shape of a globular embryo and undergo the developmental stages of embryogeny or it may develop into callus depending on the species. Regeneration of plants from pollen callus or pollen embryos may occur on the original medium or it may require transfer to a different medium. The pollen embryo exhibit considerable similarity with zygotic embryos in their morphology and certain biochemical features.
  


• Often the pollen embryos do not germinate normally. Pollen embryos frequently produce secondary embryos on stem surface and all such embryos which produce secondary embryos are haploid and the others non-haploid. To raise full plantlets from pollen embryos it is necessary to excise a cluster of the secondary embryos along with a part of the parent embryo and plant them on fresh medium. They do not germinate if left on the pollen embryo or removed individually.