Planta
Planta 137, 299-301 (1977)
9 by Springer-Verlag 1977
The Influence of Relative Humidity on the Swelling of Pollen Grains in vitro L.J.W. Gilissen Dept. of Botany, Faculty of Sciences, University, Toernooiveld, Nijmegen, The Netherlands
Abstract. The volume of hydrated pollen grains of
Petunia hybrida L. during swelling in germination medium increased three times. The volume of desiccated pollen grains increased only two times after transfer to the same medium. This difference in swelling ability is attributed to different rigidities of the pollen grain wall, caused by the different hydration states. The relationship between pollen grain swelling and germination metabolism with regard to relative humidity is discussed.
Key words: Germination (pollen) - Petunia - Pollen grain - Swelling ability.
Introduction
The germination of pollen in germination medium is greatly influenced by the relative humidity (RH) at which the pollen is pretreated. In Petunia, for example, the germination capacity of pollen showed a linear relationship with the RH at which it was equilibrated (Gilissen, unpublished). After transfer into germination medium pollen grains take up a large amount of water within a few seconds. This paper describes the effect of R H during storage of pollen grains of Petunia hybrida L. on the swelling ability of these grains in germination medium. Materials and Methods 1. Pollen
Collection of pollen of Petunia hybrida L. and storage were described previously (Gilissen, 1976, 1977). Abbreviation." RH = relative humidity
2. Relative Humidity (RH)
Equilibration of pollen at different RH's was done in air in desiccators above P2Os powder ( R H = 0 % ) , saturated solutions of CaClz (RH=35%), Ca(NO3)2 (RH=55%), (NHg)zSO4 ( R H = 8 0 % ) or above tap water (RH=100%) at room temperature. The pollen was put in open plastic tubes of 3 ml, which were layed down on a metal gauze, separating the objects from the drying powder or the solvents.
3. Pollen Diameter Measurements
The diameter of pollen grains swollen in germination medium was measured with a projection microscope (Reichert) within 45 rain. The units, which are given in the histograms, correspond with the diameter of the pollen grains in ram, as they were projected on the screen of the microscope, 1 unit corresponding to 1.67 ~tm. Every measurement consisted of 100 pollen grains.
Results
1. Influence of Relative Humidity
Figure 1 shows the diameters of swollen pollen grains of four clones after equilibration at R H = 1 0 0 % or R H = 0 % . After swelling, the diameters of hydrated and desiccated pollen grains of the clones W43, W166H and W166K followed a normal distribution. No such distribution was found for pollen grains of clone T2U. About 30% of the pollen grains of this clone appeared to be aborted (small and empty). Hydrated pollen of all four clones swoll more than desiccated pollen. Figure 2 shows the relationship between RH and swelling ability of pollen grains of clone W166K. Swelling ability increased in a linear fashion with increasing RH's from about 35% and on. The volume of a pollen grain in air was approximated by the formula 4/3 ~z abc (Fig. 3). In clone
L.J.W. Gilissen : The Influence of Humidity on the Swelling of Pollen
300
W1601(
w,3
RH=100~
10
15
20
10
25
15
20
25 diameter [unitst
Fig.3. Schematical diagram of a pollen grain in air (left) and a pollen grain after swelling in vitro (right)
T2U
W165H
2"E~
t
II
Table 1. Calculated volumes (V) of pollen grains of clones W166K and W43
Clone 10
15
20
10
25
25
20
V in air (pm 3)
V after swelling (pro 3) pollen desiccated pollen hydrated
25 diameter (units)
Fig. 1. The influence of hydration and desiccation of pollen grains of four clones of Petunia hybrida on their swelling in vitro
W 166K W43
5,400 6,200
12,800 12,000
18,500 20,200
diameter (units)
diameter (units)
20
1B
/
/
/
/
i
17 [ 0
~ 25
i
50
E 75
I 100
0
20
Z~0
retative humidity (%]
Fig. 2. The influence of the hydration state of pollen grains of Petunia hybrida clone W166K on their swelling in vitro
60
80
100
120
140 160 time (rain)
Fig. 4. The influence of time of hydration or desiccation of desiccated or hydrated pollen grains of Petunia hybrida clone W43 on
their swelling in vitro
W166K mean values of a, b and c were 17.0, 8.7 and 8.7 pro, and in clone W43 17.4, 9.2 and 9.2 gm respectively. The volume of a swollen pollen grain in germination medium was calculated according to the formula 4/3 n r 3 (Fig. 3) in which r = r a d i u s of the pollen grain. When desiccated pollen grains of clones W166K and W43 were swollen in germination
medium their mean radii became 14.5 and 14.2 gm respectively. When hydrated pollen grains of the same clones were swollen these values were 16.4 and 16.9 pm respectively. The calculated volumes are given in Table 1. It appeared that hydrated pollen grains took up much more water than desiccated pollen grains did.
L.J.W. Gilissen: The Influence of Humidity on the Swelling of Pollen
2. Duration of Hydration and Desiccation Pollen grains of clone W43 were equilibrated at R H = 0% before transfer to R H = 100%. At distinct times after transfer diameter measurements were carried out in germination medium (see Materials and Methods). Immediately following transfer to R H = 1 0 0 % the ability of the pollen grains to swell increased rapidly during the first 40 min (Fig. 4). When the opposite treatment was carried out, pollen of clone W43 was equilibrated at R H = 100% before transfer to R H = 0%, the ability to swell remained constant for about 15 min and than rapidly declined to become constant after 2 h.
Discussion
A rapid decrease in the swelling ability of pollen grains of Petunia hybrida L. was observed after transfer of the pollen from high to low RH. A similar result was obtained with regard to the germination capacity. Germination of pollen grains in germination medium (10% sucrose, 0.01% H3BO3) was minimal after pretreatment of pollen at RH = 0 % and maximal after pretreatment at R H = 100%. The low germination capacity at R H = 0% could (as could the swelling ability, see Fig. 4) wholly be restored after transfer of the pollen to R H = 100% (Gilissen, unpublished). It is suggested that the pollen grain wall becomes more rigid the more it is desiccated. This rigidity probably inhibits normal stretching of the pollen grain wall during swelling. Coster et al. (1976) have determined, that the measure of stretching of the cell wall influenced the thickness of the plasma membrane. The influence of RH on the plasma membrane of Petunia pollen grains was confirmed by diameter measurements in distilled water, which is osmotically hypotonic with regard to the pollen plasm. It appeared that pollen hydrated at R H = 3 5 % and more showed increasing pollen rupture, in contrast to desiccated pollen, which showed no rupture at all (Gilissen, un-
301
published results). The thickness of the plasma membrane, which is dependent on the stretching of the cell, influenced the cell's osmoregulation (Coster et al., 1976). Probably this osmoregulation may in turn influence the germination metabolism in the pollen protoplasm. It is also possible, however, that the germination metabolism of the pollen grain needs a special plasmawater-ratio to start functioning. In partially stretched cells, e.g. when desiccated pollen grains are put in germination medium, this ratio may be not reached and the pollen grains do not germinate. It appeared that water molecules from the air are able to penetrate and leave the pollen wall and the pollen protoplasm very easily. After penetration into the pollen grain water molecules do activate pollen respiration (Hoekstra and Bruinsma, 1975). Germination probably only occurs when enough water is taken in during the swelling of the pollen grain. This swelling ability may be dependent on uptake of water molecules from the air in a similar way as is pollen respiration. The author is much indebted to prof. H.F. Linskens for his stimulating interest, to J. van Straten (Dept. of Microbiology, Agricultural University, Wageningen) for correcting the manuscript and to Miss J. Verhoeven for typing the manuscript. Special thanks are due to Mrs. C. Polman-Molenaar, who did the initial work of this research. This investigation was made possible by financial support from the Euratom-ITAL-association, contract SC 002-09471-1 BIA N.
References Coster, H.G.L., Steudle, E., Zimmermann, U.: Turgor pressure sensing in plant cell membranes. Plant Physiol. 58, 636 643 (1976) Gilissen, L.J.W. : The role of the style as a sense-organ in relation to wilting of the flower. Planta 131,201 202 (1976) Gilissen, L.J.W. : Style-controlled wilting of the flower. Planta 133, 275 280 (1977) Hoekstra, F.A., Bruinsma, J. : Respiration and vitality of binucleate and trinucleate pollen. Physiol. Plant. 34, 221-225 (1975)
Received 20 September; accepted 3 October 1977
Planta 137, 299-301 (1977)
9 by Springer-Verlag 1977
The Influence of Relative Humidity on the Swelling of Pollen Grains in vitro L.J.W. Gilissen Dept. of Botany, Faculty of Sciences, University, Toernooiveld, Nijmegen, The Netherlands
Abstract. The volume of hydrated pollen grains of
Petunia hybrida L. during swelling in germination medium increased three times. The volume of desiccated pollen grains increased only two times after transfer to the same medium. This difference in swelling ability is attributed to different rigidities of the pollen grain wall, caused by the different hydration states. The relationship between pollen grain swelling and germination metabolism with regard to relative humidity is discussed.
Key words: Germination (pollen) - Petunia - Pollen grain - Swelling ability.
Introduction
The germination of pollen in germination medium is greatly influenced by the relative humidity (RH) at which the pollen is pretreated. In Petunia, for example, the germination capacity of pollen showed a linear relationship with the RH at which it was equilibrated (Gilissen, unpublished). After transfer into germination medium pollen grains take up a large amount of water within a few seconds. This paper describes the effect of R H during storage of pollen grains of Petunia hybrida L. on the swelling ability of these grains in germination medium. Materials and Methods 1. Pollen
Collection of pollen of Petunia hybrida L. and storage were described previously (Gilissen, 1976, 1977). Abbreviation." RH = relative humidity
2. Relative Humidity (RH)
Equilibration of pollen at different RH's was done in air in desiccators above P2Os powder ( R H = 0 % ) , saturated solutions of CaClz (RH=35%), Ca(NO3)2 (RH=55%), (NHg)zSO4 ( R H = 8 0 % ) or above tap water (RH=100%) at room temperature. The pollen was put in open plastic tubes of 3 ml, which were layed down on a metal gauze, separating the objects from the drying powder or the solvents.
3. Pollen Diameter Measurements
The diameter of pollen grains swollen in germination medium was measured with a projection microscope (Reichert) within 45 rain. The units, which are given in the histograms, correspond with the diameter of the pollen grains in ram, as they were projected on the screen of the microscope, 1 unit corresponding to 1.67 ~tm. Every measurement consisted of 100 pollen grains.
Results
1. Influence of Relative Humidity
Figure 1 shows the diameters of swollen pollen grains of four clones after equilibration at R H = 1 0 0 % or R H = 0 % . After swelling, the diameters of hydrated and desiccated pollen grains of the clones W43, W166H and W166K followed a normal distribution. No such distribution was found for pollen grains of clone T2U. About 30% of the pollen grains of this clone appeared to be aborted (small and empty). Hydrated pollen of all four clones swoll more than desiccated pollen. Figure 2 shows the relationship between RH and swelling ability of pollen grains of clone W166K. Swelling ability increased in a linear fashion with increasing RH's from about 35% and on. The volume of a pollen grain in air was approximated by the formula 4/3 ~z abc (Fig. 3). In clone
L.J.W. Gilissen : The Influence of Humidity on the Swelling of Pollen
300
W1601(
w,3
RH=100~
10
15
20
10
25
15
20
25 diameter [unitst
Fig.3. Schematical diagram of a pollen grain in air (left) and a pollen grain after swelling in vitro (right)
T2U
W165H
2"E~
t
II
Table 1. Calculated volumes (V) of pollen grains of clones W166K and W43
Clone 10
15
20
10
25
25
20
V in air (pm 3)
V after swelling (pro 3) pollen desiccated pollen hydrated
25 diameter (units)
Fig. 1. The influence of hydration and desiccation of pollen grains of four clones of Petunia hybrida on their swelling in vitro
W 166K W43
5,400 6,200
12,800 12,000
18,500 20,200
diameter (units)
diameter (units)
20
1B
/
/
/
/
i
17 [ 0
~ 25
i
50
E 75
I 100
0
20
Z~0
retative humidity (%]
Fig. 2. The influence of the hydration state of pollen grains of Petunia hybrida clone W166K on their swelling in vitro
60
80
100
120
140 160 time (rain)
Fig. 4. The influence of time of hydration or desiccation of desiccated or hydrated pollen grains of Petunia hybrida clone W43 on
their swelling in vitro
W166K mean values of a, b and c were 17.0, 8.7 and 8.7 pro, and in clone W43 17.4, 9.2 and 9.2 gm respectively. The volume of a swollen pollen grain in germination medium was calculated according to the formula 4/3 n r 3 (Fig. 3) in which r = r a d i u s of the pollen grain. When desiccated pollen grains of clones W166K and W43 were swollen in germination
medium their mean radii became 14.5 and 14.2 gm respectively. When hydrated pollen grains of the same clones were swollen these values were 16.4 and 16.9 pm respectively. The calculated volumes are given in Table 1. It appeared that hydrated pollen grains took up much more water than desiccated pollen grains did.
L.J.W. Gilissen: The Influence of Humidity on the Swelling of Pollen
2. Duration of Hydration and Desiccation Pollen grains of clone W43 were equilibrated at R H = 0% before transfer to R H = 100%. At distinct times after transfer diameter measurements were carried out in germination medium (see Materials and Methods). Immediately following transfer to R H = 1 0 0 % the ability of the pollen grains to swell increased rapidly during the first 40 min (Fig. 4). When the opposite treatment was carried out, pollen of clone W43 was equilibrated at R H = 100% before transfer to R H = 0%, the ability to swell remained constant for about 15 min and than rapidly declined to become constant after 2 h.
Discussion
A rapid decrease in the swelling ability of pollen grains of Petunia hybrida L. was observed after transfer of the pollen from high to low RH. A similar result was obtained with regard to the germination capacity. Germination of pollen grains in germination medium (10% sucrose, 0.01% H3BO3) was minimal after pretreatment of pollen at RH = 0 % and maximal after pretreatment at R H = 100%. The low germination capacity at R H = 0% could (as could the swelling ability, see Fig. 4) wholly be restored after transfer of the pollen to R H = 100% (Gilissen, unpublished). It is suggested that the pollen grain wall becomes more rigid the more it is desiccated. This rigidity probably inhibits normal stretching of the pollen grain wall during swelling. Coster et al. (1976) have determined, that the measure of stretching of the cell wall influenced the thickness of the plasma membrane. The influence of RH on the plasma membrane of Petunia pollen grains was confirmed by diameter measurements in distilled water, which is osmotically hypotonic with regard to the pollen plasm. It appeared that pollen hydrated at R H = 3 5 % and more showed increasing pollen rupture, in contrast to desiccated pollen, which showed no rupture at all (Gilissen, un-
301
published results). The thickness of the plasma membrane, which is dependent on the stretching of the cell, influenced the cell's osmoregulation (Coster et al., 1976). Probably this osmoregulation may in turn influence the germination metabolism in the pollen protoplasm. It is also possible, however, that the germination metabolism of the pollen grain needs a special plasmawater-ratio to start functioning. In partially stretched cells, e.g. when desiccated pollen grains are put in germination medium, this ratio may be not reached and the pollen grains do not germinate. It appeared that water molecules from the air are able to penetrate and leave the pollen wall and the pollen protoplasm very easily. After penetration into the pollen grain water molecules do activate pollen respiration (Hoekstra and Bruinsma, 1975). Germination probably only occurs when enough water is taken in during the swelling of the pollen grain. This swelling ability may be dependent on uptake of water molecules from the air in a similar way as is pollen respiration. The author is much indebted to prof. H.F. Linskens for his stimulating interest, to J. van Straten (Dept. of Microbiology, Agricultural University, Wageningen) for correcting the manuscript and to Miss J. Verhoeven for typing the manuscript. Special thanks are due to Mrs. C. Polman-Molenaar, who did the initial work of this research. This investigation was made possible by financial support from the Euratom-ITAL-association, contract SC 002-09471-1 BIA N.
References Coster, H.G.L., Steudle, E., Zimmermann, U.: Turgor pressure sensing in plant cell membranes. Plant Physiol. 58, 636 643 (1976) Gilissen, L.J.W. : The role of the style as a sense-organ in relation to wilting of the flower. Planta 131,201 202 (1976) Gilissen, L.J.W. : Style-controlled wilting of the flower. Planta 133, 275 280 (1977) Hoekstra, F.A., Bruinsma, J. : Respiration and vitality of binucleate and trinucleate pollen. Physiol. Plant. 34, 221-225 (1975)
Received 20 September; accepted 3 October 1977
