Planta
Planta (1988) 173:161-171
9 Springer-Verlag 1988
Growth of anthers in Lilium longifforum A kinematic analysis
Kevin S. Gould* and Elizabeth M. Lord Department of Botany and Plant Sciences, University of California, Riverside, CA 92521, USA
Abstract. The post-initiation growth of 64 anthers (1.1-17.4 mm long) in Lilium longiJlorum Thunb. was examined by time-lapse marking experiments in combination with serial sections and the scanning electron microscope. Each anther was characterized by spatial and temporal variation in growth rate. Larger anthers had two, and occasionally three, series of peaks and troughs in local growth rate. Regions of negative growth rate were frequently encountered. When observed over several days, the growth maxima and minima were found to move basipetally as a waveform down the length of the anther. The wavelength was longer in taller anthers; amplitude and frequency were variable, and anthers of the same size were not always synchronous. Distribution patterns of cell division (and elongation, once division has ceased) recapitulate the growth data. Anther growth is a non-steady system, therefore, with growth centers constantly shifting. Implications for future studies in organ growth patterns are discussed.
Key words: Anther development- Growth analysis Liliurn - Mitosis.
Introduction
There are several hundred accounts, spanning more than a century, on the initiation and histogenesis of anthers (e.g. Coulter and Chamberlain 1903; Maheshwari 1950; Davis 1966; Sattler 1973); however, none attempts to provide a complete picture of the three-dimensional growth of an anther from initiation to maturity. Most have relied on transverse or longitudinal sections, in * P r e s e n t a d d r e s s : Department of Botany, University of Auckland, Private Bag, Auckland, New Zealand Abbreviation:
SEM = scanning electron microscope
combination with direct observation or scanningelectron-microscope (SEM) images, of anther primordia. For example, Boke (1949) examined the density of cells in median longitudinal sections through stamen primordia of Vinca rosea. He concluded that until it is 200 gm tall, the stamen grows predominantly through the activity of apical and sub-apical cell initials. Thereafter, and up to its mature length of 2.5 mm, growth is intercalary. Boke considered his results supportive of the theory that stamens are homologues of leaves. The exclusive use of a limited number of sections to obtain information on the sites of meristematic activity has been criticized (Poethig 1984). Such an approach (i) confines attention to limited regions, and thereby emphasizes the role of localized meristems; (ii) restricts observations to one or two planes; and (iii) should be used only for indeterminate organs having a regular pattern of cell division, such as roots (Poethig 1984). For example, Avery (1933) studied the early development of the tobacco leaf through transverse section. He proposed that apical growth is superseded by intercalary growth before the leaf reaches 1% of its final length, and that the lamina arises from discrete marginal and sub-marginal meristems. These contentions have been refuted by many workers using an assortment of alternative techniques, including surface-marking experiments (Richards and Kavanagh 1943; Hara 1957; Maksymowych and Erickson 1960; Dubuc-Lebreux and Sattler 1980; Poethig and Sussex 1985). Until now, no surface-marking techniques have been employed to study floral organ growth. We describe here the results of a surface-growth analysis of anthers of the Easter lily, Lilium longiJTorum Thunb. Surface-marking experiments have been shown to provide an accurate description of both the spatial and material aspects of plant de-
162
K.S. Gould and E.M. Lord: Surface-growth analysis of anthers
v e l o p m e n t (Silk 1984), a n d f o r s e v e r a l r e a s o n s lily a n t h e r s are p a r t i c u l a r l y u s e f u l f o r this k i n d o f s t u d y : (i) T h e y are e x c e p t i o n a l l y l o n g (at least 25 m m a t m a t u r i t y ) a n d are t h e r e f o r e accessible to s u p e r f i c i a l m a r k i n g . (ii) B u d l e n g t h a n d a n t h e r l e n g t h are t i g h t l y c o r r e l a t e d ( E r i c k s o n 1948). T h e length of a n a n t h e r can therefore be predicted prior t o o p e n i n g u p the b u d . (iii) T h e l e n g t h o f t h e a n t h e r serves as a n a c c u r a t e i n d e x o f its d e v e l o p m e n t a l stage. E r i c k s o n (1948) s h o w e d t h a t the c y t o l o g i c a l e v e n t s l e a d i n g to m i c r o s p o r e p r o d u c t i o n o c c u r at p r e d i c t a b l e a n t h e r l e n g t h s . T h i s c h a r a c t e r i s t i c has already been utilized in physiological studies ( H o t t a a n d S t e r n 1961, 1963). (iv) T h e o n t o g e n y a n d h i s t o l o g y o f f l o r a l o r g a n s h a s b e e n well d o c u m e n t e d for the L i l i a c e a e ( G u e r i n 1927; Pfeiffer 1935; E m s w e l l e r a n d P r y o r 1943; K o s u g i 1952; D a v i s 1966; E i n e r t et al. 1970; C r e m e r et al. 1974; D e H e r t o g h et al. 1976). We present a concept of anther growth that c o u l d n o t h a v e b e e n d e m o n s t r a t e d u s i n g classical m i c r o t e c h n i q u e , a u t o r a d i o g r a p h y , or any other procedure which presupposes a steady distribution of meristems.
leum jelly had been applied over the damaged surfaces of the tepals. Photographs were taken on Kodak Technical Pan black and white film (Eastman Kodak, Rochester, N.Y., USA) and developed for maximum contrast. Prints were enlarged to a final magnification of 40 x. The distances between recognizable features on consecutive marks were measured to the nearest 0.5 mm from the enlargements using a ruler. A running refit analysis (e.g. Erickson 1976; Hunt 1982) was undertaken, using overlapping sets of four consecutive points as the measured distances. This procedure had a smoothing-out effect on the data and, since the distance between four points on the enlargements was at least 20 ram, potential errors in measurement were reduced to 5% or less. Any increment in the distance between four points, when divided by the original distance, gave a measure of the mean relative rate of elongation for that segment of the anther. Relative growth rate of each region was plotted against the distance from the base of the anther (the median distance for the original set of four points).
Material and methods Bulbs of Lilium longiflorum cv. Nellie White (Dahlstram & Watt Bulb Farms, Smith River, Cal. USA) were planted in pots and grown in a greenhouse in Riverside, Cal., until floral buds were formed. The time to flowering varied with the duration of cold storage prior to planting (Emsweller and Pryor 1943). At least two weeks before harvesting, pots were transferred to a growth chamber at 25 ~ C with a photon fluence rate of 1 mmol (photons)-m -2-s 1 and an 18 h photoperiod.
Growth in situ. The method of Erickson (1948) was used to estimate the natural growth rate of anthers in undamaged buds. Buds were harvested and dissected, and the lengths of tepals and anthers determined with a ruler. Tepal length was plotted against anther length for 52 buds (7.5-127.0 mm tong). The tepals of a further 10 buds (each borne on a different plant) were measured over 13 consecutive days. The relative rate of elongation of anthers was estimated as the product of the altometric constant k for anther and tepal lengths, and r, the mean relative rate of elongation of the tepals.
Sections. The six anthers in each of three control buds (anther lengths 0.65, 1.1, and 2.1 mm) were fixed in 2.5% glutaraldehyde buffered in potassium phosphate at pH 7.0 (Glauert 1975). They were dehydrated with 2,2-dimethoxypropane, infiltrated with and embedded in glycol methacrylate, serial-sectioned at 4 pm, and stained with 1% toluidine blue in 0.1% sodium borate (O'Brien and McCully 1981). Five of the anthers per bud were serial-sectioned transversely. Sections were scored for nuclei undergoing mitosis by making a camera-lucida drawing of the anther outline and the dividing cells. Because of the relative rarity of metaphases, cells at any stage from mid-prophase to late telophase were included. Coordinates were given to each dividing cell by placing a grid over the camera-lucida outlines. By this method successive sections through the same dividing cell could be located, so that each cell was scored for mitosis only once. Every section through anthers of the smallest bud was examined; for anthers of the two larger buds, every fifth section was examined. The sixth anther per bud, and also larger anthers processed by an identical microtechnique, were sectioned longitudinally. From approximately median longitudinal sections, the stage at which cell division ceased, and lengths of cells in different tissues of the anther were determined.
Scanning electron microscopy. Some anthers were fixed and dehydrated as above, critical-point-dried with COz, coated with gold-palladium, and viewed in a JEOL (Tokyo, Japan) 35C SEM. Others were immersed fresh into liquid nitrogen which had been supercooled under vacuum. The frozen samples were sublimated and sputter-coated with gold in an EMSCOPE (Ashford, UK) SP2000 cryo-transfer unit, and viewed in a Philips (Eindhoven, The Netherlands) 515 SEM at -170 ~ C.
Marking experiments. A small portion of the perianth was removed with a scalpel to expose the dorsal locules of one anther in situ. One of the exposed locuIes was marked with a file of approximately equidistant dots of activated charcoal in a viscous mixture with water. The first and last marks were always located at the base and tip of the locule, respectively. In total, 64 anthers varying in length from 1.1 to 17.4 mm were marked, one anther per plant. Each exposed anther was photographed alongside a reference scale. The bud was covered with polyethylene film to minimize desiccation, allowed to grow for 24 h, and then re-photographed. The 24-h period was the minimum required for measurable growth. Beyond 24 h growth was arrested unless petro-
Results Anther growth in situ. E l o n g a t i o n o f the t e p a l s is e x p o n e n t i a l i n Lilium (Fig. 1 a ) ; the r e l a t i v e r a t e o f e l o n g a t i o n , r = 0.103- d - 1. T h e r e l a t i o n s h i p bet w e e n t e p a l a n d a n t h e r l e n g t h s is b i p h a s i c (Fig. l b), y i e l d i n g a l l o m e t r i c c o n s t a n t s k = 1 . 1 4 5 a t the e a r l i e r p h a s e , a n d k = 0 . 2 5 8 b e y o n d t h e 3 5 - m m t e p a l ( 2 0 - m m a n t h e r ) stage. F r o m these
K.S. Gould and E.M. Lord: Surface-growth analysis of anthers
i63
I00
b
25
B0
[]
E C.,. (~ 15
60
r-
iO
e'-
e'-
e-
7
.._J
5
40 c 'fo
n 25' 0
2
4
6
B
10
;o
14
12
;5
Time (days)
2o 3o so 7o Length of bud (mm)
ioo
14o
Fig. 1 a, b. In-situ growth of lily floral buds. a Time course of elongation of tepals, b Logarithmic plot of anther length against tepal length O. t 0
0.t51
t'-
11o
~15
i.o
2'.5
3.0
0.I(
0.07 0.06
4~ C_
o'.s
0.0.~
0,05
0.0~
.r-~
co
O.OC
O.OZ"
CD
c~z
0.02 0.01
-0.051
i
~
3
~
~
6
7
0
i'0
15
DLstance from base of anther (mm) Fig. 2a-d. Growth profiles of individual lily anthers from surface-marking experiments. Initial anther lengths (ram): a 1.11; b 3.29; e 6.82; d 13.88
data, the relative rates of anther elongation were calculated:
kr = 0.118" d - 1 at the earlier stage, and kr = 0.027. d - 1 at the later stage. These compare with Erickson's (1948) values of kr=0.088 at the earlier stage, and kr=0.011 later on.
Marking experiments. Of the 64 anthers examined, 20 (original length 1.5-14.9 ram) grew at, or above, the calculated relative rate in situ, and 20 (2.4-17.4 ram) grew at 50-99% of the normal rate. Despite this variation, the growth profiles of 58 (91%) of the 64 anthers, including all of the 20 that grew at the normal rate, were similar. (Those of the remaining six anthers, ranging from
164
K.S. Gould and E.M. Lord: Surface-growth analysis of anthers
Table 1. Summary of data from marking experiment on anthers of Lilium longiJlorum. Arrows denote overall basipetal movement of the peak Anther length (mm)
Number of anthers Total
Trend not obvious
Numbers of peaks per anther
%Distance ~ombase at which tallest peak occurred
1
2
3
0 25
2~50
1 2 1~ 4 4 ~''-/~'-~ 5 t 1
1 / 4 2 ~
0 2.9 3 5.9 6~8.9 9-11.9 12-14.9 15-17.9
9 13 17 12 11 2
0 0 2 2 2 0
7 9 8 4 4 1
2 3 5 6 3 1
0 1 2 0 2 0
Total
64
6
33
20
5
14
O.t5
Table 2. Maximum height of the peak in growth rate of Lilium anthers from each size class and developmental stage. Mean values do not differ significantly (analysis of variance; P > 0.05) Anther size class (mm)
Developmental stage
0 2.9
Maximum height of peak (mm-mm 1-(24 h) -1) Mean
SD
Range
Mitosis in all tissues
0.19
0.08
0.09-0.31
3-5.9
Mitosis ceases in sporogenous tissues
0.23
0.11
0.0%0.35
6-8.9
Mitosis ceases in walls
0.16
0.05
0.06~.25
%11.9 12-14.9 15-17.9
0.12
Pollen mother cell meiosis
0.16
0.05
0.06-0.24
Cell elongation until maturity
0.14
0.08
0.07-0.27
0.15
0.08
0.09 0.21
6.3-14.9 m m long, fluctuated erratically; there was no discernible trend). Each anther was characterized by spatial variat i o n in r e l a t i v e g r o w t h r a t e ( F i g . 2 a - d ) , w i t h a t least one discrete region growing particularly rapidly. T a l l e r a n t h e r s h a d t w o o r t h r e e series o f p e a k s a n d t r o u g h s ( T a b l e 1 ; F i g . 2 d). The location of the growth peak varied from a n t h e r t o a n t h e r ( T a b l e 1). I t o c c u r r e d m o r e frequently nearer the tip of smaller anthers, and at the base of larger ones; however, the results were sufficiently variable to warrant an hypothesis other t h a n t h a t o f a s i m p l e d e v e l o p m e n t a l shift f r o m a p i cal to basal growth. T h e h e i g h t o f e a c h p e a k w a s v a r i a b l e ( T a b l e 2). It was neither a function of the original or final anther length, nor of a particular developmental s t a g e ( T a b l e 2). I n 40 o u t o f 64 a n t h e r s e x a m i n e d ( r a n g i n g f r o m 2.9 t o 1 5 . 5 m m in l e n g t h ) , t h e
0.0.c
1 2 1
11
~
51 75
7~100
6~"-'" 3~ 7 3~ 2 0 18
,...-3 3 2 0 0 15
Y i-2
O.OE
W
0.03
-...1c~j
0.00
.
-0.03
E E
0.08
5
I0
15
5
tO
t5
I
E cO .r-~ ('O O3 CO CD
O CLJ
0.06 0.04 0.02 0.00 -0.02
r
0.09
>
o.o8
0.07
DAY 3 - 4
~
'~
r'r O.06 0.05
0.0,~ 0.0
5
iO
Distance from base of anther
15
(mm)
Fig. 3a-e. Qrowth profiles of a lily anther, originally 12.91 mm long, over four consecutive days. Arrows denote positions of
minimum growth rate
K.S. Gould and E.M. Lord: Surface-growth analysis o f anthers
165
Fig. 4. Scanning-electron-microscope image of criticaly-point-dried lily stamens. Ventral view, anthers 1.2 mm tall. A, anther; F, filament. Bar--0.5 mm; x 36 Fig. 5. Photograph of a mature lily stamen with 25-mm anther, showing elongate dorsal locules. C, connective tissue; L, locule. Bar = 5.0 mm; x 2.2 Fig. 6. Transverse section through one locule of a lily anther 0.65 mm tall. P1 primary parietal tissue; S1 primary sporogenous tissue. Bar=0.05 ram; x 330
growth minimum fell below a value of zero (e.g. Fig. 2b, d). Regions of negative growth rate (shrinkage) partially counterbalanced regions of maximal growth, so that elongation of the anther as a whole was often considerably less than that of some of its component regions. There was a highly significant increase in peak width with increasing anther length (analysis of variance of linear regression; P
Planta (1988) 173:161-171
9 Springer-Verlag 1988
Growth of anthers in Lilium longifforum A kinematic analysis
Kevin S. Gould* and Elizabeth M. Lord Department of Botany and Plant Sciences, University of California, Riverside, CA 92521, USA
Abstract. The post-initiation growth of 64 anthers (1.1-17.4 mm long) in Lilium longiJlorum Thunb. was examined by time-lapse marking experiments in combination with serial sections and the scanning electron microscope. Each anther was characterized by spatial and temporal variation in growth rate. Larger anthers had two, and occasionally three, series of peaks and troughs in local growth rate. Regions of negative growth rate were frequently encountered. When observed over several days, the growth maxima and minima were found to move basipetally as a waveform down the length of the anther. The wavelength was longer in taller anthers; amplitude and frequency were variable, and anthers of the same size were not always synchronous. Distribution patterns of cell division (and elongation, once division has ceased) recapitulate the growth data. Anther growth is a non-steady system, therefore, with growth centers constantly shifting. Implications for future studies in organ growth patterns are discussed.
Key words: Anther development- Growth analysis Liliurn - Mitosis.
Introduction
There are several hundred accounts, spanning more than a century, on the initiation and histogenesis of anthers (e.g. Coulter and Chamberlain 1903; Maheshwari 1950; Davis 1966; Sattler 1973); however, none attempts to provide a complete picture of the three-dimensional growth of an anther from initiation to maturity. Most have relied on transverse or longitudinal sections, in * P r e s e n t a d d r e s s : Department of Botany, University of Auckland, Private Bag, Auckland, New Zealand Abbreviation:
SEM = scanning electron microscope
combination with direct observation or scanningelectron-microscope (SEM) images, of anther primordia. For example, Boke (1949) examined the density of cells in median longitudinal sections through stamen primordia of Vinca rosea. He concluded that until it is 200 gm tall, the stamen grows predominantly through the activity of apical and sub-apical cell initials. Thereafter, and up to its mature length of 2.5 mm, growth is intercalary. Boke considered his results supportive of the theory that stamens are homologues of leaves. The exclusive use of a limited number of sections to obtain information on the sites of meristematic activity has been criticized (Poethig 1984). Such an approach (i) confines attention to limited regions, and thereby emphasizes the role of localized meristems; (ii) restricts observations to one or two planes; and (iii) should be used only for indeterminate organs having a regular pattern of cell division, such as roots (Poethig 1984). For example, Avery (1933) studied the early development of the tobacco leaf through transverse section. He proposed that apical growth is superseded by intercalary growth before the leaf reaches 1% of its final length, and that the lamina arises from discrete marginal and sub-marginal meristems. These contentions have been refuted by many workers using an assortment of alternative techniques, including surface-marking experiments (Richards and Kavanagh 1943; Hara 1957; Maksymowych and Erickson 1960; Dubuc-Lebreux and Sattler 1980; Poethig and Sussex 1985). Until now, no surface-marking techniques have been employed to study floral organ growth. We describe here the results of a surface-growth analysis of anthers of the Easter lily, Lilium longiJTorum Thunb. Surface-marking experiments have been shown to provide an accurate description of both the spatial and material aspects of plant de-
162
K.S. Gould and E.M. Lord: Surface-growth analysis of anthers
v e l o p m e n t (Silk 1984), a n d f o r s e v e r a l r e a s o n s lily a n t h e r s are p a r t i c u l a r l y u s e f u l f o r this k i n d o f s t u d y : (i) T h e y are e x c e p t i o n a l l y l o n g (at least 25 m m a t m a t u r i t y ) a n d are t h e r e f o r e accessible to s u p e r f i c i a l m a r k i n g . (ii) B u d l e n g t h a n d a n t h e r l e n g t h are t i g h t l y c o r r e l a t e d ( E r i c k s o n 1948). T h e length of a n a n t h e r can therefore be predicted prior t o o p e n i n g u p the b u d . (iii) T h e l e n g t h o f t h e a n t h e r serves as a n a c c u r a t e i n d e x o f its d e v e l o p m e n t a l stage. E r i c k s o n (1948) s h o w e d t h a t the c y t o l o g i c a l e v e n t s l e a d i n g to m i c r o s p o r e p r o d u c t i o n o c c u r at p r e d i c t a b l e a n t h e r l e n g t h s . T h i s c h a r a c t e r i s t i c has already been utilized in physiological studies ( H o t t a a n d S t e r n 1961, 1963). (iv) T h e o n t o g e n y a n d h i s t o l o g y o f f l o r a l o r g a n s h a s b e e n well d o c u m e n t e d for the L i l i a c e a e ( G u e r i n 1927; Pfeiffer 1935; E m s w e l l e r a n d P r y o r 1943; K o s u g i 1952; D a v i s 1966; E i n e r t et al. 1970; C r e m e r et al. 1974; D e H e r t o g h et al. 1976). We present a concept of anther growth that c o u l d n o t h a v e b e e n d e m o n s t r a t e d u s i n g classical m i c r o t e c h n i q u e , a u t o r a d i o g r a p h y , or any other procedure which presupposes a steady distribution of meristems.
leum jelly had been applied over the damaged surfaces of the tepals. Photographs were taken on Kodak Technical Pan black and white film (Eastman Kodak, Rochester, N.Y., USA) and developed for maximum contrast. Prints were enlarged to a final magnification of 40 x. The distances between recognizable features on consecutive marks were measured to the nearest 0.5 mm from the enlargements using a ruler. A running refit analysis (e.g. Erickson 1976; Hunt 1982) was undertaken, using overlapping sets of four consecutive points as the measured distances. This procedure had a smoothing-out effect on the data and, since the distance between four points on the enlargements was at least 20 ram, potential errors in measurement were reduced to 5% or less. Any increment in the distance between four points, when divided by the original distance, gave a measure of the mean relative rate of elongation for that segment of the anther. Relative growth rate of each region was plotted against the distance from the base of the anther (the median distance for the original set of four points).
Material and methods Bulbs of Lilium longiflorum cv. Nellie White (Dahlstram & Watt Bulb Farms, Smith River, Cal. USA) were planted in pots and grown in a greenhouse in Riverside, Cal., until floral buds were formed. The time to flowering varied with the duration of cold storage prior to planting (Emsweller and Pryor 1943). At least two weeks before harvesting, pots were transferred to a growth chamber at 25 ~ C with a photon fluence rate of 1 mmol (photons)-m -2-s 1 and an 18 h photoperiod.
Growth in situ. The method of Erickson (1948) was used to estimate the natural growth rate of anthers in undamaged buds. Buds were harvested and dissected, and the lengths of tepals and anthers determined with a ruler. Tepal length was plotted against anther length for 52 buds (7.5-127.0 mm tong). The tepals of a further 10 buds (each borne on a different plant) were measured over 13 consecutive days. The relative rate of elongation of anthers was estimated as the product of the altometric constant k for anther and tepal lengths, and r, the mean relative rate of elongation of the tepals.
Sections. The six anthers in each of three control buds (anther lengths 0.65, 1.1, and 2.1 mm) were fixed in 2.5% glutaraldehyde buffered in potassium phosphate at pH 7.0 (Glauert 1975). They were dehydrated with 2,2-dimethoxypropane, infiltrated with and embedded in glycol methacrylate, serial-sectioned at 4 pm, and stained with 1% toluidine blue in 0.1% sodium borate (O'Brien and McCully 1981). Five of the anthers per bud were serial-sectioned transversely. Sections were scored for nuclei undergoing mitosis by making a camera-lucida drawing of the anther outline and the dividing cells. Because of the relative rarity of metaphases, cells at any stage from mid-prophase to late telophase were included. Coordinates were given to each dividing cell by placing a grid over the camera-lucida outlines. By this method successive sections through the same dividing cell could be located, so that each cell was scored for mitosis only once. Every section through anthers of the smallest bud was examined; for anthers of the two larger buds, every fifth section was examined. The sixth anther per bud, and also larger anthers processed by an identical microtechnique, were sectioned longitudinally. From approximately median longitudinal sections, the stage at which cell division ceased, and lengths of cells in different tissues of the anther were determined.
Scanning electron microscopy. Some anthers were fixed and dehydrated as above, critical-point-dried with COz, coated with gold-palladium, and viewed in a JEOL (Tokyo, Japan) 35C SEM. Others were immersed fresh into liquid nitrogen which had been supercooled under vacuum. The frozen samples were sublimated and sputter-coated with gold in an EMSCOPE (Ashford, UK) SP2000 cryo-transfer unit, and viewed in a Philips (Eindhoven, The Netherlands) 515 SEM at -170 ~ C.
Marking experiments. A small portion of the perianth was removed with a scalpel to expose the dorsal locules of one anther in situ. One of the exposed locuIes was marked with a file of approximately equidistant dots of activated charcoal in a viscous mixture with water. The first and last marks were always located at the base and tip of the locule, respectively. In total, 64 anthers varying in length from 1.1 to 17.4 mm were marked, one anther per plant. Each exposed anther was photographed alongside a reference scale. The bud was covered with polyethylene film to minimize desiccation, allowed to grow for 24 h, and then re-photographed. The 24-h period was the minimum required for measurable growth. Beyond 24 h growth was arrested unless petro-
Results Anther growth in situ. E l o n g a t i o n o f the t e p a l s is e x p o n e n t i a l i n Lilium (Fig. 1 a ) ; the r e l a t i v e r a t e o f e l o n g a t i o n , r = 0.103- d - 1. T h e r e l a t i o n s h i p bet w e e n t e p a l a n d a n t h e r l e n g t h s is b i p h a s i c (Fig. l b), y i e l d i n g a l l o m e t r i c c o n s t a n t s k = 1 . 1 4 5 a t the e a r l i e r p h a s e , a n d k = 0 . 2 5 8 b e y o n d t h e 3 5 - m m t e p a l ( 2 0 - m m a n t h e r ) stage. F r o m these
K.S. Gould and E.M. Lord: Surface-growth analysis of anthers
i63
I00
b
25
B0
[]
E C.,. (~ 15
60
r-
iO
e'-
e'-
e-
7
.._J
5
40 c 'fo
n 25' 0
2
4
6
B
10
;o
14
12
;5
Time (days)
2o 3o so 7o Length of bud (mm)
ioo
14o
Fig. 1 a, b. In-situ growth of lily floral buds. a Time course of elongation of tepals, b Logarithmic plot of anther length against tepal length O. t 0
0.t51
t'-
11o
~15
i.o
2'.5
3.0
0.I(
0.07 0.06
4~ C_
o'.s
0.0.~
0,05
0.0~
.r-~
co
O.OC
O.OZ"
CD
c~z
0.02 0.01
-0.051
i
~
3
~
~
6
7
0
i'0
15
DLstance from base of anther (mm) Fig. 2a-d. Growth profiles of individual lily anthers from surface-marking experiments. Initial anther lengths (ram): a 1.11; b 3.29; e 6.82; d 13.88
data, the relative rates of anther elongation were calculated:
kr = 0.118" d - 1 at the earlier stage, and kr = 0.027. d - 1 at the later stage. These compare with Erickson's (1948) values of kr=0.088 at the earlier stage, and kr=0.011 later on.
Marking experiments. Of the 64 anthers examined, 20 (original length 1.5-14.9 ram) grew at, or above, the calculated relative rate in situ, and 20 (2.4-17.4 ram) grew at 50-99% of the normal rate. Despite this variation, the growth profiles of 58 (91%) of the 64 anthers, including all of the 20 that grew at the normal rate, were similar. (Those of the remaining six anthers, ranging from
164
K.S. Gould and E.M. Lord: Surface-growth analysis of anthers
Table 1. Summary of data from marking experiment on anthers of Lilium longiJlorum. Arrows denote overall basipetal movement of the peak Anther length (mm)
Number of anthers Total
Trend not obvious
Numbers of peaks per anther
%Distance ~ombase at which tallest peak occurred
1
2
3
0 25
2~50
1 2 1~ 4 4 ~''-/~'-~ 5 t 1
1 / 4 2 ~
0 2.9 3 5.9 6~8.9 9-11.9 12-14.9 15-17.9
9 13 17 12 11 2
0 0 2 2 2 0
7 9 8 4 4 1
2 3 5 6 3 1
0 1 2 0 2 0
Total
64
6
33
20
5
14
O.t5
Table 2. Maximum height of the peak in growth rate of Lilium anthers from each size class and developmental stage. Mean values do not differ significantly (analysis of variance; P > 0.05) Anther size class (mm)
Developmental stage
0 2.9
Maximum height of peak (mm-mm 1-(24 h) -1) Mean
SD
Range
Mitosis in all tissues
0.19
0.08
0.09-0.31
3-5.9
Mitosis ceases in sporogenous tissues
0.23
0.11
0.0%0.35
6-8.9
Mitosis ceases in walls
0.16
0.05
0.06~.25
%11.9 12-14.9 15-17.9
0.12
Pollen mother cell meiosis
0.16
0.05
0.06-0.24
Cell elongation until maturity
0.14
0.08
0.07-0.27
0.15
0.08
0.09 0.21
6.3-14.9 m m long, fluctuated erratically; there was no discernible trend). Each anther was characterized by spatial variat i o n in r e l a t i v e g r o w t h r a t e ( F i g . 2 a - d ) , w i t h a t least one discrete region growing particularly rapidly. T a l l e r a n t h e r s h a d t w o o r t h r e e series o f p e a k s a n d t r o u g h s ( T a b l e 1 ; F i g . 2 d). The location of the growth peak varied from a n t h e r t o a n t h e r ( T a b l e 1). I t o c c u r r e d m o r e frequently nearer the tip of smaller anthers, and at the base of larger ones; however, the results were sufficiently variable to warrant an hypothesis other t h a n t h a t o f a s i m p l e d e v e l o p m e n t a l shift f r o m a p i cal to basal growth. T h e h e i g h t o f e a c h p e a k w a s v a r i a b l e ( T a b l e 2). It was neither a function of the original or final anther length, nor of a particular developmental s t a g e ( T a b l e 2). I n 40 o u t o f 64 a n t h e r s e x a m i n e d ( r a n g i n g f r o m 2.9 t o 1 5 . 5 m m in l e n g t h ) , t h e
0.0.c
1 2 1
11
~
51 75
7~100
6~"-'" 3~ 7 3~ 2 0 18
,...-3 3 2 0 0 15
Y i-2
O.OE
W
0.03
-...1c~j
0.00
.
-0.03
E E
0.08
5
I0
15
5
tO
t5
I
E cO .r-~ ('O O3 CO CD
O CLJ
0.06 0.04 0.02 0.00 -0.02
r
0.09
>
o.o8
0.07
DAY 3 - 4
~
'~
r'r O.06 0.05
0.0,~ 0.0
5
iO
Distance from base of anther
15
(mm)
Fig. 3a-e. Qrowth profiles of a lily anther, originally 12.91 mm long, over four consecutive days. Arrows denote positions of
minimum growth rate
K.S. Gould and E.M. Lord: Surface-growth analysis o f anthers
165
Fig. 4. Scanning-electron-microscope image of criticaly-point-dried lily stamens. Ventral view, anthers 1.2 mm tall. A, anther; F, filament. Bar--0.5 mm; x 36 Fig. 5. Photograph of a mature lily stamen with 25-mm anther, showing elongate dorsal locules. C, connective tissue; L, locule. Bar = 5.0 mm; x 2.2 Fig. 6. Transverse section through one locule of a lily anther 0.65 mm tall. P1 primary parietal tissue; S1 primary sporogenous tissue. Bar=0.05 ram; x 330
growth minimum fell below a value of zero (e.g. Fig. 2b, d). Regions of negative growth rate (shrinkage) partially counterbalanced regions of maximal growth, so that elongation of the anther as a whole was often considerably less than that of some of its component regions. There was a highly significant increase in peak width with increasing anther length (analysis of variance of linear regression; P
