Pollen Physiology Palynophysiology

Pollen physiology has attracted the attention of plant breeders and horticulturists for plant improvement programmes ever since the discovery of the pollen tube by Giovanni Batista Amici (1924), an Italian astronomer and mathematician, while examining the papillate stigma of Portulaca oleracea. Physiological studies have mainly centred round pollen germination, storage, and on the artificial induction of pollen sterility in cultivated plants, to be of benefit in large-scale hybridization programmes. Very scanty information is available in pollen germination of Indian trees. An interesting development in the pollen studies is the study of the influence of pollen physiology on the mode of pollination and breeding systems of plants. In pollen physiology, the various aspects normally studied are: pollen chemistry, pollen storage, pollen viability, pollen germination in vitro and in vivo.

POLLEN GERMINATION

Each mature pollen grain upon germination produces two nuclei by mitotic division of the haploid nucleus of the dehisced grain. One of these nuclei (vegetative) controls the growth of the pollen tube as it progresses down the stylar tissue, the remaining nucleus (gametic) follows down the tube as it grown towards the micropyle of the ovule, at which point the gametic nucleus bypasses the vegetative one, and enters the micropyle to fuse with the haploid ovule nucleus, thus restoring the diploid state with regard to chromosome and gene complement, to form the zygote from which develops the embryo plant upon seed germination (Fig. 10.1).

The arrival of a pollen grain upon the stigmatic surface of the gyno-ecium (female part of the flower) is stimulated by chemicals secreted by the stigmatic tissues (usually a sugar compound) to produce the pollen tube via one or more of the exine pores.

Pollen Tube Calcium

Successful pollination and fertilization are essential for sexual reproduction in higher plants. The germination of pollen requires a varying range of growth media like water, sugars, inorganic salts, hormones and vitamins. However, some species are known to germinate in distilled water without addition of any nutrients.

As the studies of pollen germination in vivo are difficult, our knowledge of the physiology and biochemistry of pollen germination and pollen tube growth is based on in vitro germination studies. In addition to moisture, carbohydrates, boron and calcium are generally essential prerequisites for artificial (in vitro) pollen germination. Pollen grains of most of the species require an optimum concentration of sugar solution and sucrose is the best carbohydrate source for pollen germination and tube growth.

Extract from flower parts, growth substances like kinetin, gibberellic acid, indole compounds, etc. and even some antibiotics (e.g. penicillin) are known to have a stimulating effect on pollen germination and tube growth. The temperature and pH have a profound effect on germination of the pollen grains. Ideally a temperature of 20°C to 30°C and a pH range of 5.5 to 7.0 have been found to be optimum for pollen germination.

The time interval between pollen hydration and tube initiation is termed as the lag-phase. The duration of the lag-phase varies from species to species. In some species it is limited to a few minutes while in others it may extend to many hours. The pollen tube emerges through the germ pore. It generally emerges as an extension of the intine. The intine, therefore, has to become less rigid before the pollen tube can emerge. One of the special features of the pollen tube is that growth is confined to the tip only.

Pollen grains of different species of plants have specific requirements for their germination. In some cases pollen germinate in ordinary tap water, starch paste with one or two parts of water and on parchment paper soaked with sugar solution or even in moist air. Following these observations, attempts have been made to cultivate pollen in vitro. Cane sugars have been frequently employed as a medium with a good rate of success. Various kinds of sugars (alone or with agar), nutrient elements (both major and minor) and some growth substances and vitamins have been used in germinating pollen grains of different species of plants.

Pollen germination studies involve assessment of the role of various substances mentioned below:

1. Role of sugars (nutritive)

2. Role of boron (a stimulant of pollen germination and pollen tube growth).

3. Effect of certain growth substances (like Indole Acetic Acid).

4. Role of lipids

Various steps are involved in the process of pollen germination. Pollen hydration, germination and penetration of the stigma by pollen tubes are influenced by the exudate on wet stigmas and by the pollen wall in species with dry stigmas. The exudate is known to allow pollen tubes to grow directly into the stigma, whereas the pollen wall establishes the contact with the stigma. This is followed by the pollen tubes growing into the papillae, which are covered by a cuticle.

Wolters-Arts et al. (1998) while working on the nature and role of exudate in tobacco plants, showed that lipids are the essential factor needed for pollen tubes to penetrate the stigma. They further concluded that lipids in the exudate direct pollen tube growth by controlling the flow of water to pollen in species with dry and wet stigmas.

METHOD OF STUDYING IN VIVO GERMINATION OF POLLEN GRAINS

In vivo germination of pollen grains in hybrids and their respective female parents is studied on the first, second and third day after opening of the flower. In the above experiment, the stigmas along with some portion of the styles are taken out of the flowers after the first, second and third day of anthesis. They were then subjected to the following treatments:

1. At first the stigmas along with part of style are kept in aceto-alcohol (acetic acid:alcohol 1:1) at 60°C for one and half hour.

2. They are washed with distilled water and macerated in 1% KOH solution at 60°C for an hour.

3. The stigmas were washed with distilled water and kept in it at 60°C for one hour and then again washed twice or thrice with water.

4. After washing, the stigmas are kept at 30°C in lactic acid for 10 minutes.

5. They are then stained in cotton blue. Slides are prepared by mounting the material in glycerine by exerting sufficient pressure and examined under the microscope for assessing in vivo germination of pollen

According to Heslop-Harrison et al. (1973) proteins are stored at two sites in pollen grain walls: in exine derived from tapetum, and intine synthesized by pollen grain itself during the course of development. These observations were made in Malvales. Such sporophytic and gametophytic fractions of pollen wall proteins are supposed to play an important role in pollen stigma interaction and they are, therefore, mostly concerned with compatibility reactions (Heslop-Harrison 1971).

As pollen grains come in contact with the stigma, it is soon subjected to hydration. Simultaneously the pollen wall proteins are released on the stigma, first the exine proteins and then the entire proteins (Knox and Heslop-Harrison 1975a).

Pollen grains are usually shed under dehydrated conditions (watercontent <20%), and the metabolic rate is very low (Wilson et al., 1979) and it is expected that endogenous substrates would be gradually used up. Stored pollen grains require a higher sugar concentration for germination in vitro than fresh pollen (Johri and Vasil 1961).

POLLEN VIABILITY

Pollen grains contain the male gamete and may be regarded as microspores. Their viability is relatively short as the cytoplasmic contents have limited food reserves to maintain the living state, and as with all gametes, both plant and animal, viability is designed to be maintained to cover transfer and pollination.

Environmental factors, particularly temperature and humidity, greatly affect pollen viability. Retention of the pollen viability after shedding is termed as pollen longevity, and varies significantly from species to species. There is a close correlation between the cytology of pollen (two or three-celled) and loss of viability. Two-celled pollen grains generally retain viability for a longer period, than three-celled pollen grains (Brewbaker 1957). Two-celled pollen grain is more amenable to storage compared to three-celled pollen.

The most commonly used method for assessing pollen viability is in vitro germination of the pollen grains. It is rapid, reasonably simple, and fully quantitative. The percentage of in vitro germination can be correlated with its ability to set fruits and seeds following in vivo pollination (Visser 1955; Akihama et al., 1978). Whereas in in vivo conditions, the most authentic and accurate test for viability is expressed as the ability of pollen to effect fertilization resulting in seed and fruit set. This fruit and seed set test has many limitations (Heslop-Harrison et al, 1984). It is laborious and more time consuming.

METHODS OF DETERMINATION OF POLLEN VIABILITY

Triphenyl tetrazolium chloride (TTC) reacts with viable pollen and gives red colouration. Tetrazolium chloride and its derivatives have been used as vital stains for testing viability of tissues especially in seeds. Triphenyl tetrazolium chloride (TTC) is commonly known as tetrazolium salt. It is a white to pale yellow crystalline powder that darkens on exposure to light and is readily soluble in water.

In the presence of viable tissue the colourless solutions of 2, 3, 5-triphenyl tetrazolium chloride forms the insoluble red triphenyl formazon. This reduction reaction is caused by the dehydrogenase enzymes present in living tissues.

The TTC stain is kept in 10% stock solutions and diluted to the ratio - 1 part of stain: 5 parts of 60% sucrose solution at the time of treatment. However, a disadvantage of sucrose is the possibility that it may become contaminated with bacteria or other microorganisms and be decomposed by them. To guard against this contamination the two solutions should be kept separately until the slides are prepared.

Procedure

A drop of the final dilution is placed on a slide, and pollen from just dehisced anthers are dusted on the surface and a cover slip is placed on it. Two hours are required for the development of red colour. The proportion of grains developing a red colour was determined by counts made with a microscope using the low power objective, after 2 hours. The percentage of coloured grains is estimated. Coloured grains were considered viable and grains without colour were considered non viable. Thus, the pollen at the known time of anthesis can be tested for viability after several days.

METHODS FOR TESTING GERMINATION OF STORED POLLEN IN VITRO FOR VIABILITY OF STORED POLLEN

The anthers are collected in Petri dishes and allowed to dehisce in the laboratory. The whole pollen sample was divided equally into required number of small lots, transferred to small glass vials, which were loosely closed with cotton plugs and kept at the following temperatures and humidity combinations:

At room temperature 25°C - 29°C with 0, 5, 10, 25 or 30% RH

At freeze temperature (9°C - 12°C) with 0, 5, 10, 25 or 30% RH

The humidity was maintained in desiccators by a mixture of sulphuric acid and water. The viability tests of stored pollen are carried out at intervals of 24 hours. Viability is tested by germinating the pollen grains in vitro, in castor oil, which is found to be the best medium for germination of cotton pollen.

For this, pollen grains just after anthesis are sown on a drop of refined castor oil and spread on a microslide in the form of a circle, 1-5 cm in diameter. The pollen grains are carefully stirred so that the pollen grains are coated with oil completely. The slide with the pollen is placed on a stand in a Petri dish in which there is a thin layer of water. The dish is partly covered to allow free circulation of air and kept in total darkness at 38 c for 24 hours. The germination percentage and tube lengths are recorded after 24 hours.

Pollen Storage

As an aspect of conservation, the technique of preserving shortlived gametes-pollen and spores - by means of freeze-drying is used. This technique involves storing, samples of pollen in small glass vials, which are then placed in containers of liquid nitrogen. A state of 'suspended animation' ensues throughout the period of storage. Upon gradual return to normal temperatures, the pollen may be persuaded to fertilize female ovules, which like pollen can receive similar freeze-drying. Pollen banks have a role to play in the possible production of species on the verge of extinction.

Successful pollen storage is a very convenient tool in the hands of tree breeders for improving trees by hybridization, occurring in different regions and also of those blooming in different seasons. The longevity of pollen grains enables the introduction of new characters in plants by using stored living pollen. Thus, there is an immense importance of pollen storage in tree improvement programmes.

Jain and Shivanna et al. (1990) and other scientists have successfully stored pollen grains of several taxa in organic solvents such as benzene, petroleum ether, ethanol, acetone and chloroform. Certain pollen diluents have been found to be very effective in increasing pollen longevity. Powdered egg albumin, stale and powdered milk are useful in storing pollen as dry and powdery diluents.

The pollen grains are often mixed with diluents such as wheat flour, corn flour, powdered charcoal, talc and Lycopodium spores before storage (Johri and Vasil 1961). The main purpose of diluents is to increase the bulk of pollen so as to avoid wastage of the pollen sample during artificial pollination. Pollen grains of many taxa have been successfully stored in ultra-low temperature using dry ice (-80°C) or liquid nitrogen (-196°C), in suitable cylinders. The refrigerant has to be replenished at regular intervals. This technique is known as Freeze-Drying or Lyophilization. Generally, a mixture of solid CO2 and acetone is used for freezing.

Lower temperature and humidity (0°-8°C and 0-50 RH) prolong the life of pollen grains of different plants (Johri and Vasil 1961; King, 1965; Holman and Brubaker 1926). Cross-pollination largely depends on insects. Poor weather conditions limit the activity of insects, and significantly reduce the yield because of insufficient pollination. To overcome this problem, attempts were made to carry manual pollinations with the help of stored pollen (Brown and Perking 1969; Legge 1976; Williams and Legge 1979). In the U.S.A. and U.K. there are several commercial firms, which sell stored pollen for supplementary pollinations. The ultimate aim of storage of the pollen grains by plant breeders is to establish 'pollen banks' through which the viable pollen of the desired species can be obtained at any time. Pollen banks are often established for the supply of viable pollen for mass scale hybridization. Pollen storage of viable pollen in pollen banks is done by various methods, such as storage by controlling temperature and humidity, storage by freezing and dehydration, influence of gases and pressure, storage with pollen diluents.

POLLEN CRYOBANK AT THE INDIAN INSTITUTE OF HORTICULTURAL RESEARCH (IIHR), BANGALORE, INDIA

Long term cold storage (cryopreservation) of nuclear genetic diversity in the form of pollen in several plants of horticultural, medicinal and forest importance can be successfully achieved (Ganeshan et al., (2005). The ultimate result of this methodology is the establishment of the Pollen Cryobank at the Indian Institute of Horticultural Research in Bangalore, India which maintains more than 600 types of pollen belonging to 45 species and 15 families under cryogenic conditions.

Sustainable use of cryopreserved nuclear genetic diversity has been successfully demonstrated through field pollination with cryopreserved pollen. Thus, the cryopreserved pollen from the pollen bank can be put to use by plant breeders, hybrid seed producers, genebanks for various crops of economic importance. The IIHR maintains in the database, information pertaining to longevity, viability and media used for pollen germination, yearwise listing of collected pollen samples and yearwise lists of pollen samples cryopreserved in different years. Some of these cryopreserved pollen in the pollen bank maintained by the pollen storage laboratory of the Division of Plant Genetic Resources, IIHR includes pollen of Day lily, mango, citrus, Carnation, Gladiolus, Vinca rosea, grape, papaya, pomegranate, tomato, lemon, onion, rose, Solanums and capsicum.

Successful storage of pollen grains of some tree species by manipulation of temperature and humidity is shown in Table 10.1 (courtesy Sharma).

Table 10.1 Successful storage of the pollen grains of some tree species by manipulation of temperature and humidity.

Temperature (°C)

Relative humidity (%)

Duration of storage

References

Artocarpus sp.

0

25

8 months

Sinha 1972

Betula sp.

5

0 (vacuum)

2.5 years

Jensen 1964

Juglans nigra

4

-

3 months

Hall and

Farmer 1971

Mangifera indica

0

0

14 months

Singh 1960

Pinus banksiana

2

25-75

1 year

Johnson 1943

P.nigra

5

0 (vacuum)

2.5 years

Jensen 1964

P.strobes

18

25

14 months

Duffield and

Snow 1941

P.resinosa

0-4

25-50

14 months

Duffield and

Snow 1941

P.sylvestris

2

25-75

1 year

Johnson 1943

Prunus americana

2-8

50

2.5 years

Nebel and

Ruttle 1937

P.communis

2

0

1.5 years

King and

Hesse 1938

Pyrus communis

2

0

3 years

Visser 1955

Populus sp.

18

Desiccated

Many years

Knox et al.

over silica gel

1972 b.

for 12-16 h before storage for 12-16 h before storage

The self-incompatibility is considered to be one of the main causes for the rapid evolution of angiosperms. Pollen is known to have a higher energy investment per gram of organic tissue than other plant parts. Pollen of anemophilous spp. have lower caloric contents than pollen of entomophilous species. In addition to its main function of pollination and fertilization, pollen attracts and nurtures a variety of pollinators. Enhanced pollen energy contents could attract pollen consumers for their energy requirements.

Though physiological studies have been undertaken mainly on pollen germination, the biochemical and biophysical factors responsible for successful germination are not as yet clearly understood.

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Responses

  • Magnus
    How shedding viability of pollengrains depends on humidity and temperature?
    2 years ago
  • degna
    What effect has sugar has on pollen grain?
    2 years ago
  • jessamine
    Which indian flower to test pollain germination?
    2 years ago
  • Eeva Ahokas
    How chemical is affect pollen tube germination?
    1 year ago
  • adelbert
    How are pollen grains stored ?
    1 year ago
  • demi-lee
    What the function of following substance in the pollen grain germinating solution?
    11 months ago
  • belladonna
    Why carbohydrates important for pollen germination?
    7 months ago
  • diana
    How are different varities of pollen grains stored for long period of time in pollen banks?
    7 months ago
  • Matthias
    How we grow pollen tube without using boron?
    6 months ago
  • Ailey
    Why does a pollen grain need sugar to grow?
    6 months ago

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