Table Of ContentERGEBNISSE
DER BIOLOGIE
HERAUSGEGEBEN VON
H. AUTRUM . E. BONNING . K. v. FRISCH
E. HADORN . A. KOHN . E. MAYR . A. PIRSON
J. STRAUB . H. STUBBE· W. WEIDEL
REDIGIERT VON
HANSJOCHEM AUTRUM
DREIUNDZWANZIGSTER BAND
MIT 40 ABBILDUNGEN
SPRINGER-VERLAG
BERLIN· GOTTINGEN • HEIDELBERG
1960
ISBN-13: 978-3-640-02611-6 e-ISBN-IS: 978-3-642-94770-4
Dor: 10.1007/978-3-642'94770-4
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Inhaltsverzeichnis
The Pollen and Pollen Tube. By Dr. B. M. JOHRI and Dr. I. K. VASIL, Delhi
(India) .............................. .
Vergleichende Biochemie der Phenylpropane. Von Privatdozent Dr. H. REZNIK,
Heidelberg. Mit 5 Abbildungen. . . . . . . . . . . . . . . . . . . . . 14
Photomorphogenetische Reaktionssysteme in Pflanzen. 2. Teil: Der EinfluB
kurzwelligen Lichts auf Wachstum und Entwicklung. Von Professor Dr. H.
MOHR, Tiibingen. Mit 18 Abbildungen. . . . . . . . . . . . . . . . . . 47
Homing Orientation in Migrating Fishes. By Professor Dr. A. D. HASLER,
Madison 6, Wis. (USA). With 6 Figures. . . . . . . . . . . . . . . . . 94
Beziehungen zwischen Entomophagen und ihrer Beute als Grundlage der bio-
logischen Schadlingskontrolle. Von Dr. R. LANGE, Freiburg i. Br. Mit 1 Ab-
bildung. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
Die Biologie der kondensierten anorganischen Phosphate. Von Dr. A. KUHL,
GOttingen. Mit 2 Abbildungen ..................... 144
Dber die Rolle der Frequenzgruppe beim Horen. Von Dozent Dr. E. ZWICKER,
Stuttgart. Mit 8 Abbildungen . . . . . . . . . . . . . . . . . . . . . 187
Namenverzeichnis. 204
Sachverzeichnis .. 217
The Pollen and Pollen Tube*
By B. M. J OHRI and I. K. VASIL
Department of Botany, University of Delhi, Delhi 6, India
Contents
Storage of Pollen · 2
Germination of Pollen · 4
Literature · 9
In 1694, CAMERARIUS reported that anthers play the role of male
sexual organs in plants (MAHESHWARI, 1950). Later, AMICI observed
pollen tubes travelling through the stigma and style of Portulaca oleracea
and in 1830 he concluded that pollen tubes elongate and ultimately
come in contact with the ovule (MAHESHWARI, 1950). Since then the
work of numerous investigators has shown that the pollen tube carries
two male gametes into the embryo sac where one of them fuses with
the egg which finally forms the embryo while the other gamete fuses
with the polars or secondary nucleus which gives rise to the endosperm.
Under natural conditions, pollen grains of most plants lose their
viability in a short time after shedding. In Hordeum (ANTHONY and
HARLAN, 1920) and Oryza (NAGAO and TAKANO, 1938) fertilization
usually does not occur unless the pollen is directly transferred from the
anthers to the stigma and seeds could not be obtained in Sorghum
(STEPHENS and QUINBY, 1934) with five-hour old pollen. On the contrary,
pollen of certain fruit trees and gymnosperms is reported to retain
viability for long periods (HOLMAN and BRUBAKER, 1926; NEBEL and
RUTILE, 1937; VISSER, 1955).
Attempts of plant breeders and horticulturists to produce new
varieties have often been failed due to certain difficulties, e.g. difference
in time or place of flowering of the parents, pollen grains not germinating
on the stigma, and slow growth or death of pollen tubes in the style
(BLAKESLEE, 1945; MAHESHWARI, 1950). Sometimes, when the tubes are
able to reach ovules, sterility results either due to failure of the male
gamete to fuse with the egg or the subsequent failure of development
of the embryo and endosperm. Recent researches have helped to overcome
* This paper is the result of a programme of work financed by the Indian
Council of Agricultural Research, New Delhi.
Ergebnisse der Biologie XXIII
2 B. M. J ORRI and 1. K. VASIL
some of these hurdles and the use of viable stored pollen has proved of
great advantage. The relevant literature on the storage and viability of
pollen has been reviewed by HOLMAN and BRUBAKER (1926), NEBEL
and RUTTLE (1937) and more recently by VISSER (1955).
Storage of pollen is of immense value in plant breeding schemes.
The transport of pollen is not controlled by strict plant quarantine
measures as applied to the transport of live plants or seeds. In the
U.S.A. commercial firms (ANTLES, 1951) regularly sell stored pollen
to orchardists for artificial pollinations. Apart from its importance for
practical purposes, successful pollen storage is also useful for physio-
logical studies.
Storage of Pollen
The earliest reference (2,000 B.C.) on the handling and storage of
pollen concerns the pollen grains of date palm. According to POPENOE,
pollination with seven years old pollen was entirely successful (see NEBEL
and RUTTLE, 1937). However, STOUT (1924) and CRAWFORD (1937) have
doubted the validity of such statements.
Systematic research on storage can be said to have begun at the end of
the nineteenth century when several workers studied the longevity of pollen
of more than 80 species under air-dry conditions (exposed to room tempe-
rature and humidity). Thus, it was discovered that the pollen of many spe-
cies remained viable for a longer period at low temperatures and under dry
conditions (HODIAN and BRUBAKER, 1926). PFUNDT (1910) reported that
the pollen of most species retained viability for the maximum period at
low (0 and 30%) than at high (90%) relative humidity (R. H.), and that
of Prunus padus remained alive for 15 days at 90 %,22 days at 60 %, and
181 days at 30 % and 0 % R. H. His results were later confirmed by HODIAN
and BRUBAKER (1926) who concluded that "storage at low humidities
triples on the average the longevity of those pollens which it affects".
However, KNOWLTON (1922) found 50-80% R. H. to be the optimum
for the pollen of maize. NEBEL and RUTTLE (1937) stated: "it appears
that for the pollen of the species and varieties of Prunus, Pyrus and
V itis tested, a humidity of 50 % is possibly the optimum, the life curves
becoming increasingly shorter and steeper as humidity is further in-
creased and becoming shorter as humidity is further decreased". Again,
PFEIFFER (1936, 1938) has shown that there is a close correlation between
longevity and the moisture content of the atmosphere to which the
pollens are exposed, with maxima and minima of different pollen at
different humidities.
The work done in this laboratory indicates that the pollen of Brassica
nigra, Solanum melongena and S. tuberosum (]OHRI and VASIL, 1957a;
VASIL, 1958c), Arachis hypogaeaand Pennisetu.m typhoideum (VASIL, 1958a)
The Pollen and Pollen Tube 3
remains viable longest at 0 % or 31-40 % R. H. According to VISSER (1955)
the longevity of pollen is in general negatively correlated with the relative
humidity required for optimal storage. It appears that the majority of
pollen show optimum longevity at relatively low humidities and are,
therefore, drought resistant.
Pollen storage at temperatures as low as, or even below, the freezing
point has also been tried. In 1901, GOFF reported that viability of fruit
tree pollen was not impaired by exposure to sub-zero temperatures.
KNOWLTON (1922) observed that the pollen of Antirrhinum showed
maximum viability at _18° to -30° C and no reduction in its germina-
bility was noticed even when exposed for half an hour at -190° C.
At -12° C, OLl\iO (1942) was able to preserve pollen of Vitis vinifera for
1,461 days while that of Pyrus communis and P. malus kept at _17° C
remained viable for 3,287 days (USHIROZAWA and SHIBUKAWA, 1951).
The storage of pollen of several fruit trees in dry ice (solid carbon dioxide
at -55° to -60° C) has also proved very useful (GRIGGS et aI., 1950;
ANTLES, 1951). BREDEMANN et aI. (1947) stored the pollen of Lupinus
polYPhyllus at -180° C (liquid air) for 93 days, while VISSER (1955)
stored the pollen of Prunus communis, Rhododendron catawbiense and
Lycopersicum esculentum for 662 days and of Pyrus malus for 673 days
at a temperature of -190° C (liquid oxygen) with only a negligible
loss in viability.
The pollen of Brassica nigra (J OHRI and VASIL, 1957b) remains
viable for 55 days at 5-8° C (31-40% R. H.) while at room temperature
(16-20° C and 31-40% R. H.) it was viable for 34 days. That of
Solanum tuberosum is viable for 18 days at 15-31° C (31-40% R. H.)
but at 5-8° C the viability extends to 70 days (JOHRI and VASIL,
1957b). Pollen of Gossypium herbaceum, which loses viability within 24 hours
at room temperature, could be kept viable for 55 hours at -40 C (VASIL,
1958a).
In general, near-freezing temperatures and a relative humidity of
25-50% have proved most suitable for maintaining the viability for
maximum periods.
In sharp contrast to the relatively long viability of the pollens listed
above, the longevity of the pollen of Gramineae has been found to be
extremely short under all conditions so far tested. Low and intermediate
humidities (0-40% R. H.), so useful in most other cases (VISSER, 1955),
are definitely harmful (the only exception being Penniset~tm typhoideum;
see VASIL, 1958a).
Reduced air-pressure has been reported to be favourable in maintain-
ing viability for considerable periods, e.g. in Citrus (KELLERMAN, 1915),
Lilium (PFEIFFER, 1936, 1938), apple and pear (VISSER, 1955); while the
pollen of barley (ANTHONY and HARLAN, 1920), sugarcane (SARTORIS,
1*
4 B. M. J ORRI and 1. K. VASIL
1942) and Cinchona (PFEIFFER, 1944) remains viable longer at normal than
at reduced pressure. High percentages of carbon dioxide, which follow
storage over dry ice, increase the longevity (KNOWLTON, 1922; ANTLES,
1951) while storage in pure oxygen is less favourable (KNOWLTON, 1922).
OVERLEY and BULLOCK (1947), PFEIFFER (1948), and ANTLES (1951)
have studied the effect of various diluents on the storage of pollen and
it has been found that lycopodium powder, egg albumen and casein help
in prolonging viability. Diluents are believed to check desiccation and
are widely used now to increase the bulk of pollen during artificial
pollination programmes.
PFUNDT (1910) attempted correlations between the duration of
viability and the taxonomic relationships of the species studied by him.
He concluded that the more closely related the species, the greater the
agreement in the longevity of their pollen. HOLMAN and BRUBAKER
(1926) have also dealt with this question in some detail and have given
definite rankings to various families according to their pollen longevity.
Several workers have experienced that the stored pollen usually shrivels
and shows poor germination. However, it grows better if exposed to higher
humidities for some time before germination in higher concentrations
of sucrose than are required by fresh pollen (VASIL, 1958a). The probable
causes of the loss of viability may be desiccation and loss (utilization
during metabolism) of stored food. Moreover, the inactivation of enzymes
preceded by desiccation may cause failure of metabolic processes re-
sponsible for the maintenance of viability (see KNOWLTON, 1922; NEBEL
and RUTTLE, 1937; NIELSEN et aI., 1955; NIELSEN, 1956). Although
there is no detailed qualitative or quantitative analysis to demonstrate
the loss of stored food and inactivation of enzymes during storage, fairly
convincing evidences have been put forward from time to time. Quite
often stored samples of pollen do not germinate in vitro but on pollina-
tion give a satisfactory fruit set which shows that probably the losses
suffered during storage are somehow made up in vivo (KNOWLTON, 1922;
HOLMAN and BRUBAKER, 1926; STEPHENS and QUINBY, 1934; OLMO,
1942; HAGIAYA, 1949; VISSER, 1955; VASIL, 1958a).
It is now agreed that under favourable temperature and humidity
pollen of most plants remains viable for various periods. Recently,
BREDEMANN et aI. (1947) have drawn attention to the fact that the
germinating capacity of Lupinus pollen would remain unaltered for
"some millions of years" when stored at -180° C and would be virtually
"eternal".
Germination of Pollen
In 1834 VON MOHL noticed, for the first time, that in a saturated
atmosphere pollen of some plants readily produced tubes. Later, SCHLEI-
DEN (1849) and VAN TIEGHEM (1869) studied in vitro germination of
The Pollen and Pollen Tube 5
pollen of several species. Since then many workers have successfully
germinated pollen grains of numerous species in artificial media. It is
interesting to note that the percentage of germination and length of the
pollen tube varies from species to species or even from variety to variety.
It has been the experience of most workers that for successful
germination of pollen in vitro, sugar of some kind or the other is always
necessary. Generally sucrose has been used for this purpose but lactose
(BISHOP, 1949), dextrose (FAULL, 1955), and several other sugars have
also proved satisfactory (O'KELLEY, 1955; TANAKA, 1955; RAGHAVAN and
BARUAH, 1956a; JOHRI and VASIL, 1957a; VASIL, 1958b, 1960). The role
of sugar in pollen germination and pollen tube growth was not clearly
understood until recently. Some of the earlier workers believed that it
regulates the osmotic pressure of the medium and also serves as a source
of nutrition for the pollen (BRINK, 1924b; O'KELLEY, 1955). VISSER
and some others (see VISSER, 1955), on the other hand, maintain that the
function of sugar is exclusively of an osmotic nature. However, by using
labelled sugars for the germination of Tecoma radicans pollen, O'KELLEY
(1955) proved conclusively that the externally supplied sugars are
utilized in respiration. HELLMERS and MACHLIS (1956) have also established
that the pollen of pine absorbs and utilizes externally supplied carbo-
hydrates.
Apart from sugars, there are several other substances, particularly
boron (in the form of boric acid or borax), which have a marked effect
on the germination of pollen (SCHMUCKER, 1935; ANTLES, 1949, 1951;
BATJER and THOMPSON, 1949; GAUCH and DUGGAR, 1954; JOHRI and
VASIL, 1955, 1957a; VISSER, 1955; O'KELLEY, 1957; VASIL, 1958a,b, 1960).
Boron not only reduces the bursting of pollen in vitro but also increases the
percentage of germination and length of the pollen tube. As far as is
understood at present, it reacts with the sugar in the medium and
forms an ionisable sugar-borate complex which is readily available to the
pollen tube for metabolic purposes (GAUCH and DUGGAR, 1953; LINSKENS,
1955). It increases oxygen absorption (O'KELLEY, 1957) and is necessary
for cell wall formation (SPURR, 1957).
Extracts of stigma, style, ovule or anther have also been found to
be useful (KUHLWEIN and ANHAEUSSER, 1951; ANHAEUSSER, 1953; MIKI,
1954, 1955). KATZ (1926) considered the stigmatic secretion to be in-
dispensable for the successful germination of pollen but believed
that it had no chemical influence on germination. The pollen tubes of
several plants exhibit positive chemotropism to pieces of stigma, ovules
and other floral organs (TSAO, 1949; VISSER, 1955). MIKr (1954) states:
"In Camellia sinensis, pollen tubes show positive tropism to the fresh
style slices while they show negative tropism to slices from the styles
which are steamed for 10 minutes at 60, 80 or 99° C. Both substances,
6 B. M. J OHRI and 1. K. VASIL
which are responsible for the positive and negative tropism, diffuse from
styles to agar media". She further states that probably "the active factor
is a chemical substance" (See also MI KI, 1955, 1959).
The aggregation of pollen grains in culture has a striking effect both
on the percentage of germination and length of tube (BECK and ]OLY,
1941; VISSER, 1955). VISSER (1955) mentions: "The experiments with
an increasing number of grains per drop and those with pollen extracts
imply that a substance (or substances) with 'germination promoting'
properties diffuses from the grains."
BRINK (1925) demonstrated the effect of PH on the germination of
pollen of Lathynts odoratus where optimum germination was secured
between PH 6 and PH 8. He suggested that "the hydrogen-ion concentra-
tion may modify pollen tube growth through a direct effect upon the
chemical reactions attending the digestion of the reserve food materials".
BERG (1930) obtained single (Berberis aqttifolium, PH 4.5) and double
(Aesculus fiava, PH 4.2 and 7.4) optima curves for pollen germination in
PH range 2.5 to 9, although the maxima and minima differed with
different pollens.
Temperature also affects germination (VISSER, 1955) and the optimum
lies between 20° and 30° C (BERG, 1930). At low temperatures germina-
tion is considerably slow and in vivo the tubes may not elongate
sufficiently to reach the ovules and affect fertilization (SMITH and
COCHRAN, 1935). However, satisfactory germination can be obtained at
sub-optimal temperatures, provided ample time is allowed for growth.
ADAMS (1916) reported 90-100% germination in apple pollen at 14° C
after 24 hours, while at 2° C, OSTLIND (1945) obtained 10-36% and
90-100% germination after 69 and 119 hours respectively.
The diameter of pollen tubes increases with a corresponding increase
in temperature (SMITH, 1942). The pollen of Antirrhinum (SMITH, 1942)
showed negligible growth at 15° C, optimum germination and length of
tubes at 25° C, pronounced broadening and bloating of the distal portions
of tubes at 30° C, and extensive bursting of the bloated tubes at 35° C.
For a half-hour growth period, Medicago sativa (SEXSlITITH and FRYER,
1943) showed a linear relationship between pollen tube growth and rise
in temperature. SEN and VAR:I[A (1956) report that in Pismn sativum
"as compared to room temperature (10-12° C) increased germination
and elongation of pollen tubes was obtained at 15° to 21° C. At tem-
peratures below 12° C the germination was comparatively poor, while
at 24° C the pollen tubes burst profusely". In Areca catecku optimum
germination was recorded at 28° C, whereas at 15°, 20°, 300 and 35°
germination was inhibited (RAGHAVAN and BARUAH, 1956a). In Dolichos
lablab, in a range of 17.5° (room temperature) to 40° C, optimum growth
of tubes was observed at 30°, other temperatures proved inhibitory and
The Pollen and Pollen Tube 7
at higher temperatures (35-400 C) bloating and broadening of distal
portion of pollen tubes was common (VASIL, 1958a). "An increase in
temperature of 100 C approxinately doubled the relative influence of
boron on the germination rate" (VISSER, 1955). VASIL (1958a) also found
QI0 ± 2 in the case of Dolichos lablab.
Growth of pollen tube in vitro is usually much less than what would
be required in nature to affect fertilization. However, in Chionodoxa,
Hippeastrum, Muscari, Puschkinia and Scilla (BRINK, 1924b), Pachy-
phytum and Sedum (VISSER, 1955), and Crotalaria juncea (VASIL, 1958a)
the pollen tubes attain sufficient length even in vitro.
Vitamins (ADDICOTT, 1943; ANTLES, 1949; ANHAEUSSER, 1953;
SEN and VARMA, 1955, 1956), hormones (SMITH, 1942; ADDICOTT,
1943; SEN and VARMA, 1955, 1956), antibiotics, pigments and
certain other chemicals have often been reported to affect the germination
of pollen and growth of tubes. According to CURTIS and DUNCAN (1947),
orchid pollen contains enough auxin to secure normal germination,
whereas in Antirrhinum and Bryophyllum the auxin content is low and
limits germination (SMITH, 1942). COOPER (1939) considers that the
influence of several vitamins and amino acids on the germination of
Carica papaya pollen may be due to PH. BRINK (1924a) obtained a
substantial increase in the germination' of pollen of Cucumis sativ~ts,
Lythrum salicaria and Primula abconica by the addition of sterile yeast
to the culture medium. KATO (1955; see also STODOLA, 1957) accelerated
the rate of pollen tube growth in Lilium longiflorum with the help of
gibberellic acid. In Pisum sativum gibberellin not only accelerated
growth of the tubes but in some cases there was a supernumerary divi-
sion of the male gametes with the result that each tube contained four male
gametes (BOSE, 1959). LIDFORSS (1896) observed that even in small doses
sodium chloride and potassium nitrate prove toxic to pollen (TOKUGAWA,
1914; BRINK, 1924a). However, contrary to the results obtained by
these workers, several recent reports indicate that at least calcium nitrate
and manganese sulphate do improve germination (Loo and HWANG,
1944; SEN and VARMA, 1955; RAGHAVAN and BARUAH, 1956b; VASIL,
1958b, 1960). PULVERTAFT (1946) has shown that penicillin favours ger-
mination and the work of VASIL (1958b, 1960) confirms that penicillin
and streptomycin have a 'stimulatory effect on germination and tube
growth of Cucumis melo var. utilissimus and M omordica charantia.
Certain carotenoids have been reported by SCHWARZENBACH (1953) to
increase the percentage of pollen germination in Cyclamen persicum
while some others inhibit it.
Studies on the physiology of pollen are of utmost importance to
plant breeders, particularly in solving problems of incompatibility.
Numerous investigators (BATJER and THOMPSON, 1949; ANTLES, 1951;
