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In this article we will discuss about the tests that have proven to determine the viability and vigour of pollen after storage in plants.
I. Test for Pollen Viability:
An assessment of pollen viability is imperative factor in the study of reproductive biology, pollen storage and hybridization. Thus there must be a quick and dependable method to determine the viability of a pollen grain. Several tests have been standardized from time to time each with its own limitations are briefly discussed below.
i. In vitro Germination Test:
It is the most popular and quantitative, simple speedy test for assessing pollen viability A small sample of the pollen is observed under microscope and the per-cent of grains producing tubes after a given time is determined. This per-cent is considered an index of viability of the pollen sampled.
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Such tests assume that the optimum conditions have been established for the in vitro test so that germination approximates that on the plant. However, pollen tubes of most of the grains cultured in vitro stop growing before they reach the size normally attained in the style and the rate of tube growth is seldom as rapid as in vivo. This suggests that optimum growth conditions are not always established in vitro media.
Pollen grains of Antirrhinum remain viable up to 670 days, but show only 180 day of viability in vitro, this however, can be extended if a piece of stigma is placed in the medium.
This again suggests that the inability to germinate in vitro following storage due to deficiency of some substances is compensated by the stigma. Thus pollen, which loses germinability following storage, cannot be considered completely nonviable, and needs to be tested by supplementing in vivo pollination.
ii. Pollen Germination on Stigma:
By controlled pollination and observing the growth of the pollen tube through the style it is possible to some extent in assessing the viability of a pollen grain. At least 70 pollen tubes in the style of Brassica oleracea is considered to indicate total viability of the pollen grains.
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In-spite of the limitations of available techniques (fluorescent method) for observing the pollen tube through the style, it is better than the laborious and time- consuming method of fruit and seed set technique.
iii. Enzyme Assay Method:
It demonstrates cytochemically the activity of certain enzymes in living pollen, like dehydrogenases by the triphenyl tetrazolium chloride (TTC) test; catalases by the benzidine test or esterases by the fluorescein diacetate (FDA) test, etc. It has to be remembered that the presence of a functional enzyme in the cell itself does not guarantee that the pollen is still viable and functional.
iv. Fruit and Seed Set:
It is the most genuine way of testing viability and is expressed by the ability of the pollen to effect fertilization resulting in seed and fruit set. The system has many limitations like, receptivity and incompatibility of the stigma; post- fertilization determinants related to seed development may thwart seed-set/ not applicable in apomixis reproduction; tedious and time-consuming system; and applicable only at the time of flowering period and thus cannot be used as routine practice.
v. Tetrazolium Test:
The most commonly used tetrazolium salt in pollen viability test is 2, 3, 5-triphenyltretra- zolium chloride. Tetrazolium test is based on the reduction of soluble colourless tetrazolium salt to reddish insoluble formazan in the presence of dehydrogenase (Fig 7.3).
If the pollen grains are incubated in tetrazolium solution (0.1-1%) for 30-60minutes at 30-37 °C, viable pollen grains take a reddish colour due to the formation of formazan. Since the tetrazolium salts can be easily red iced by light, it is necessary to keep the solution and the pollen grains in dark.
vi. Fluorochromatic Reaction Test (FCR) or Fluorescein Diacetate Test (FDA):
The Fluorescein di-O-acetate (Fig.7.4) is a non-polar, nonfluorescent compound that can be rapidly hydrolyzed by acetyl esterase activity to fluorescein, a polar, fluorescent compound that is retained by the living cells only.
Heslop- Harrison & Heslop-Harrison (1970) introduced the FDA test for pollen viability, which assesses two properties of the pollen, viz., the integrity of the plasma membrane of vegetative cell; and the activity of esterase capable of cleaving the fluorescein di-O-acetate.
The test has been widely accepted for its better resolving power than the other existing tests of viability. Viable pollen grains when mounted in a suitable osmoticum FDA solution, non-polar, non-fluorescent FDA penetrates the plasma membrane of the vegetative cell and enters the pollen cytoplasm.
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Hydrolytic cleavage of the FDA by esterases produces fluorescein, which accumulates in the pollen cytoplasm, as the intact plasma membrane is impermeable to it. This fluorescein gives bright fluorescence when observed under fluorescent microscope (Fig. 7. 5).
In non-viable pollen the plasma membrane is generally disrupted as a result it allows the fluorescein to move out readily and produce uniform background fluorescence. Further, in the absence of active esterases in the pollen cytoplasm the fluorescein is not formed and thus pollen grains do not fluoresce.
II. Test for Pollen Vigour:
The following three tests have proven to determine the vigour of pollen:
i. In-vitro Germination:
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In-vitro germination not only assesses the viability of pollen but can also determine its vigour. If germination of stored Nicotiana tabacum pollen is recorded at intervals over an interval of time and compared with the germinability rate of fresh pollen (control pollen), it would appear that pollen grains with reduced vigour take longer time to achieve maximum germinability. (Fig. 7.6).
ii. Semi-Vivo Method:
By using unpollinated compatible pistil sample pollen grains are tested. The pistils are maintained either on the plant in an aseptic condition or are excised and maintained in the nutritive culture medium. Pistils pollinated in such a manner are incubated for 3-6 hours until pollen grains germinate on the stigma and the pollen tube grows down for some length in the style.
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After required time of incubation period the style is cut ahead of the growing pollen tubes and the cut end of the style is embedded in the agar medium containing the required nutrients for pollen germination. The pollen tubes continue their growth and invade the agar medium after emerging through the cut end of the style.
The number of emerged pollen tubes are counted, their length measured and time taken for their emergence is also noted. Pollen vigour is determined by the number of emerged pollen tubes and the time taken by the said tubes to emerge from the style.
iii. In-Vivo Pollen Germination and Pollen Tube Growth:
Vigour of sample pollen can also be determined by pollinating them on pistils along with fresh pollen and measuring the pollen tube growth at regular intervals. The growth of the vigorous sample pollen will be faster than the control pollen.
