PlantCell Reports
Plant Cell Reports (1986) 5:47-49
© Springer-Verlag 1986
Microspore growth and anther staging in barley anther culture W. G. Wheatley, A. A. Marsolais, and K. J. Kasha Department of Crop Science, University of Guelph, Guelph, Ontario, Canada, N 1G 2W1 Received July 8, 1985 / Revised version received November 18, 1985 - Communicated by F. Constabel
ABSTRACT: Nuclear growth, microspore c e l l growth and c e l l c y c l e stage were examined in microspores o f anthers o f Hordeum vulgare L. cv. Klages taken from f l o r e t s o f the middle o f the spike as per anther staging methods. Although there was wide v a r i a t i o n in nuclear size at a l l stages o f the c e l l c y c l e , mean nuclear size appeared to be a good i n d i c a t o r o f c e l l cycle stage f o r microspores w i t h i n anthers. Microspore c e l l size increased c o n s i d e r a b l y during G1 o f the c e l l c y c l e . Anthers bearing microspores c y t o l o g i c a l l y c h a r a c t e r i z e d as in the m i d - u n i n u c l e a t e stage, which have proven to y i e l d high l e v e l s o f c a l l u s p r o d u c t i o n , were determined to be in GI o f the c e l l c y c l e and were r e g u l a r l y found in spikes taken from t i l l e r s in which the base o f the f l a g l e a f had emerged 0 to 3 cm above the p e n u l t i m a t e l e a f . INTRODUCTION: In b a r l e y , anther response and c a l l u s i n d u c t i o n f o l l o w i n g anther c u l t u r e has been found to depend s i g n i f i c a n t l y on the s e l e c t i o n o f microspores at the m i d - u n i n u c l e a t e stage regardless o f whether spikes are subjected to pretreatments p r i o r to c u l t u r e (Sunderland et a l . , 1979) or anthers removed and p l a t e d direct-Ty-TMarsolais and Kasha, 1985). In o t h e r species, wide v a r i a t i o n in nuclear size has been observed f o r anthers at various stages o f microspore development (White and Davidson, 1976). Such v a r i a t i o n has led to some confusion when attempts are made to compare c y t o l o g i c a l c h a r a c t e r i s t i c s of microspores and r e l a t i v e p o s i t i o n o f the microspores in the c e l l c y c l e . While i t has been suspected t h a t s e l e c t i o n of m i d - u n i n u c l e a t e microspores in GI o f the c e l l c y c l e (Sunderland et a l . , 1979) was associated w i t h increased a n t h e r - response and p r o d u c t i v i t y , o t h e r s have suggested t h a t microspores in S and G2 o f the c e l l cycle were p r e f e r a b l e (Sun, 1978). In t h i s study, we have examined the nuclear and c e l l u l a r growth of microspores during the f i r s t c e l l c y c l e o f p o l l e n development in the b a r l e y v a r i e t y Klages which responds well to anther c u l t u r e and r e l a t e d our o b s e r v a t i o n s to anther staging f o r anther c u l t u r e . MATERIALS AND METHODS: The two-rowed, spring b a r l e y c u l t i v a r Klages (Hordeum v u l g a t e L.) was used in t h i s study. Plants were grown as p r e v i o u s l y described (Marsolais and Kasha, 1985) and developing spikes were selected from the f i r s t t i l l e r s o f the p l a n t s . P r i o r to the removal and f i x a t i o n o f each o f the spikes in a c e t i c a c i d : alcohol ( 1 : 3 ) , the d i s t a n c e from the base o f
Offprint requests to: W. G. Wheatley
the f l a g l e a f to the penultimate l e a f was measured (cm) and recorded. A f t e r at l e a s t I day, the spikes were t r a n s f e r r e d to 70% alcohol and 6 to 9 anthers removed from 2 o r 3 f l o r e t s in the middle of the s p i k e . The anthers were stained according to the method of M i t c h e l l (1967) using d i n i t r o f l o r o b e n z e n e and Feulgen s t a i n s . A f t e r s t a i n i n g , the anthers were placed on microscope s l i d e s and the microspores removed by g e n t l e tapping with a d i s s e c t i n g needle. Permanent s l i d e s were made by using the method of Conger and F a i r c h i l d (1953). Measurements of c e l l areas, nuclear areas, maximum and minimum diameters of nuclei were made through a camera l u c i d a using a Zidas d i g i t i z i n g table~(Zeiss),.Nucl~a~ vqlumes wer~ c a l c u l a t e d accorm~ng to wnl~e anm uavlmson (Tg76). At l e a s t 50 c e l l s and nuclei were measured f o r each sample. The measurements of nuclear DNA content were made on 25 nuclei from each sample using a Zeiss MPV03 scanning microspectrophotometer at a wavelength o f 560 nm. RESULTS: Microspore nuclear size varied g r e a t l y w i t h i n anthers o f each sample and over the f i r s t m i t o t i c c e l l cycle of the microspore (Figures la and i b ) . Nuclei r e c e n t l y formed from the equational d i v i s i o n of meiosis ( i . e . e a r l y GI nuclei w i t h i n c e l l s forming t e t r a d s , f i g u r e 2a) had a mean nuclear volume o f 74.2 20.9 ~m3 ~ mean nuclear area = 22.7 + 4.0 pm2) compared to nuclei in prophase ( l a t e G2) o f the f i r s t p o l l e n m i t o s i s ( f i g u r e 2d) which had a mean nuclear volume of 402.3 + 130.2 ~m3 (mean nuclear area = 71.2 + 13.4 ~m2)~ On average, the nuclei increased in size by 5 . 4 - f o l d in terms of volume o r 3 . l - f o l d in terms of area over the course of the c e l l c y c l e . Even though there was c o n s i d e r a b l e v a r i a t i o n in e i t h e r nuclear volume or nuclear area, i t was apparent t h a t the c e l l c y c l e stage of the p o p u l a t i o n of microspores taken from anthers l o c a t e d at the middle o f a spike could be estimated from the mean nuclear area and/or mean nuclear volume (Figures la and I b ) . Anthers t h a t contained microspores having a mean nuclear #olume ranging from 104.1 + 35.6 to 157.9 + 58.8 ~ma (areas = 28.7 + 5.3 to 39.4 + 8.6 pm~) were in G1 phase as estimated by DNAmeasurements (Figure 2b). Anthers c o n t a i n i n g microspores having mean nuclear volume ranging from 246.6 + 93.0 to 312.2 + 130.2 pm3 (area = 51.6 + 10.4 t o 6 1 . O + 15.3 ~m~) had i n t e r m e d i a t e DNA values which Tndicated t h a t these p o p u l a t i o n s were undergoing DNA synthesis (S phase, f i g u r e 2c).
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Figure 1: Nuclear size d i s t r i b u t i o n s and nuclear DNA amounts for microspores at various stages of the c e l l cycle. (Mean + S.D.) a) Nuclear area (pm2) versus nuclear DNA am-ount (A.U. = a r b i t r a r y u n i t s ) , b) Nuclear volume (lJm3) versus nuclear DNA amount.
Figure 2: Stages of microsporogenesls during f i r s t c e l l cycle in b a r l e y . a) Tetrad stage ( e a r l y G1, no microspore cell wall observed, small nucleus). b) Mid-uninucleate microspore in G1 ( c e l l s walls present, vacuole present and small nucleus displaced to periphery of microspore). c) Microspore in S phase ( s i m i l a r to 2b, nucleus slightly larger). d) Late-uninucleate microspore ( l a t e G2, large sperical nucleus in prophase of m i t o s i s ) . Bar denotes 10 lJm.
Tahle 1: Cell areas for c e l l s at stages during meiosis and microsporogenesis STAGE
CELL AREA (~m2) mean + S.D.
Microspores observed in the G2 phase had a mean nuclear volume of 336.4 + 118.4 j£m3 (area = 64.1 + 12.4 pm2): the mean DNA~ontent approached that the prophase n u c l e i . Part of the observed v a r i a t i o n in nuclear DNA content was due to b i r e f r i g e n c e of the microspore c e l l w a l l . In p a r t i c u l a r , t h i s contributed to the wider than expected range of mean DNA contents found in the GI microspores.
Meiotic prophase I Tetrad stage ( e a r l y G1) GI microspores G2 microspores (prophase of first mitosis)
A summary of the measurements of c e l l areas for c e l l s in prophase I of meiosis, t e t r a d stage, G1 microspores and microspores having nuclei undergoing prophase of the f i r s t pollen mitosis is presented in Table 1. Considerable v a r i a t i o n in c e l l size was observed over these stages. On average, the area of c e l l s at t e t r a d stage decreased to about 27% t h a t of c e l l s in meiotic prophase I. For microspores t h a t were in G1 of the c e l l cycle the mean c e l l area was about 5 to 6 - f o l d l a r g e r than the c e l l area of an average t e t r a d c e l l . Vacuole formation and expansion w i t h i n the developing microspores contributed g r e a t l y to t h i s increase in size. By the time of the f i r s t pollen mitosis the microspores size had increased by about 7 - f o l d compared to the t e t r a d c e l l s . Part of t h i s increase in size above the GI microspores appeared to be due to t h i c k e n i n g of the microspore cell wall.
The distance from the base of the f l a g l e a f blade to the base of the penultimate l e a f blade has been used as morphological t r a i t to assist in estimating the proper stages of the spike p r i o r to e x c i s i o n . Anthers which were removed from t i l l e r s in which the f l a g l e a f had emerged from 0 to 3 cm have proven to give the best percentage anther response f o l l o w i n g c u l t u r e (75-80% anther response, 10+15 c a l l i per responding a n t h e r ) . This observation applied to the c u l t i v a r Klages grown in our environment. These microspores were in G1 of the c e l l cylce and had comparatively small mean nuclear sizes and large vacuoles. These t r a i t s were c y t o l o g i c a l l y c h a r a c t e r i s t i c of the mid-uninucleate stage. Other observations i n d i c a t e d that spikes with anthers in which the f l a g l e a f had emerged beyond 3 cm could be
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59,1 22,3 86,1 69.0
49 in GI, S or G2 o f the c e l l cycle and that the distance of flag l e a f emergence in which the f i r s t pollen mitosis was observed tended to be from 9.5 to 12 cm, although l a t e r and e a r l i e r stages of pollen formation were also found in t h i s range. Thus, while the use of t i l l e r in which the flag l e a f had recently emerged appeared to assist the selection of spikes which had anthers at the proper stage i t was not a s t r i c t c r i t e r i o n for selection of spikes which had mid-uninucleate (GI) microspores. DISCUSSION In t h i s study, the objective was to determine the precise c e l l cycle stage associated with high levels of anther response and p r o d u c t i v i t y . C e l l u l a r parameters such as c e l l area and nuclear size were measured and compared to spectrophotometric measurements of nuclear DNA content. Microspores within barley anthers increased considerably in c e l l size over the f i r s t c e l l cycle and was due p r i m a r i l y to vacuole formation and enlargement. By comparison, nuclear growth appeared more gradual over t h i s phase of development and exhibited wide v a r i a t i o n over the course of the c e l l cycle. Precise staging could not be accomplished by estimating the nuclear size of i n d i v i d u a l microspores. However, mean nuclear size estimates of microspores populations o f 50 c e l l s derived from individual anthers d i f f e r e n t i a t e d mid-uninucleate microspores in GI from those in G2 of the cell cycle and perhaps those in S phase. We suspect that imprecise staging of microspore development and v a r i a t i o n in microspore stage along the spike contributes s i g n i f i c a n t l y to sub-optimal levels of anther response and p r o d u c t i v i t y . In general, anthers taken from f l o r e t s in the middle o f the spike tended to be in more advanced stages of microsporogenesis compared to anthers in adjacent f l o r e t s . Anthers found in f l o r e t s in the bottom and top quarters of the spike lagged considerably behind in t h e i r development compared to anthers in the middle of the spike. Our results suport the suggestion by Sunderland eft a l . (1979) that mid-uninucleate and l a t e -
uninuclcu~u microsprores d i ~ e r ir~ huclear size. Howeven, the d i s t r i b u t i o n s of nuclear size of these stages overlapped due to inherent v a r i a b i l i t y and developmental asynchrony. Based on our measurements, the mid-uninucleate stage which gives high levels of anther response and p r o d u c t i v i t y corresponds to the G1 stage of the c e l l cycle. Although Sunderland et a l . (1979) indicated that the use of morphological-c h a r a c t e r i s t i c s of the t i l l e r s was not s a t i s f a c t o r y for d i s t i n g u i s h i n g between spikes bearing mid-uninucleate and late-uninucleate microspores, the distance of flag l e a f emergence proved to be a reasonably r e l i a b l e i n d i c a t o r of microspore stage with the c u l t i v a r Klages grown in our environment. Nevertheless, we recommend that spikes should be staged i n i t i a l l y using the r e l a t i v e l y simple method of estimating mean nuclear area. These results should be correlated with morphological t r a i t s such as flag l e a f emergence that vary with genotypes and environments. Improvements in anther staging procedures should lead to increased anther response and p r o d u c t i v i t y and a decrease in the error component within experir~ents. ACKNOWLEDGEMENTS Financial support provided by the Natural Sciences and Engineering Research Council of Canada and by the Ontario Ministry of Agriculture and Food is g r a t e f u l l y acknowledged. The technical assistance of Sylvia Flack in the operation of the scanning microspectrophotometer is greatly appreciated. REFERENCES: Conger, A.D., F a i r c h i l d , L.M. (1953) Stain Tech. 28: 281-283. Marsolais, A.A., Kasha, K.J. (1985) Can. J. Bot. (accepted) M i t c h e l l , J.P. (1967) J. Roy. Microsc. Soc. 87:106-123. Sun, C.S. (1978) In: Proc. Symp. Plant Tissue Culture. Peking, China, Science Press, Peking, China, pp. 117-125. Sunderland, N., Roberts, M., Evens, L.J., Wildon. D.C. (1979) J. Exp. Bot. 30:1133-1144 White, R.L., Davidson, D. (1976) Can. J. Genetic. Cytol. 18:385-396
Plant Cell Reports (1986) 5:47-49
© Springer-Verlag 1986
Microspore growth and anther staging in barley anther culture W. G. Wheatley, A. A. Marsolais, and K. J. Kasha Department of Crop Science, University of Guelph, Guelph, Ontario, Canada, N 1G 2W1 Received July 8, 1985 / Revised version received November 18, 1985 - Communicated by F. Constabel
ABSTRACT: Nuclear growth, microspore c e l l growth and c e l l c y c l e stage were examined in microspores o f anthers o f Hordeum vulgare L. cv. Klages taken from f l o r e t s o f the middle o f the spike as per anther staging methods. Although there was wide v a r i a t i o n in nuclear size at a l l stages o f the c e l l c y c l e , mean nuclear size appeared to be a good i n d i c a t o r o f c e l l cycle stage f o r microspores w i t h i n anthers. Microspore c e l l size increased c o n s i d e r a b l y during G1 o f the c e l l c y c l e . Anthers bearing microspores c y t o l o g i c a l l y c h a r a c t e r i z e d as in the m i d - u n i n u c l e a t e stage, which have proven to y i e l d high l e v e l s o f c a l l u s p r o d u c t i o n , were determined to be in GI o f the c e l l c y c l e and were r e g u l a r l y found in spikes taken from t i l l e r s in which the base o f the f l a g l e a f had emerged 0 to 3 cm above the p e n u l t i m a t e l e a f . INTRODUCTION: In b a r l e y , anther response and c a l l u s i n d u c t i o n f o l l o w i n g anther c u l t u r e has been found to depend s i g n i f i c a n t l y on the s e l e c t i o n o f microspores at the m i d - u n i n u c l e a t e stage regardless o f whether spikes are subjected to pretreatments p r i o r to c u l t u r e (Sunderland et a l . , 1979) or anthers removed and p l a t e d direct-Ty-TMarsolais and Kasha, 1985). In o t h e r species, wide v a r i a t i o n in nuclear size has been observed f o r anthers at various stages o f microspore development (White and Davidson, 1976). Such v a r i a t i o n has led to some confusion when attempts are made to compare c y t o l o g i c a l c h a r a c t e r i s t i c s of microspores and r e l a t i v e p o s i t i o n o f the microspores in the c e l l c y c l e . While i t has been suspected t h a t s e l e c t i o n of m i d - u n i n u c l e a t e microspores in GI o f the c e l l c y c l e (Sunderland et a l . , 1979) was associated w i t h increased a n t h e r - response and p r o d u c t i v i t y , o t h e r s have suggested t h a t microspores in S and G2 o f the c e l l cycle were p r e f e r a b l e (Sun, 1978). In t h i s study, we have examined the nuclear and c e l l u l a r growth of microspores during the f i r s t c e l l c y c l e o f p o l l e n development in the b a r l e y v a r i e t y Klages which responds well to anther c u l t u r e and r e l a t e d our o b s e r v a t i o n s to anther staging f o r anther c u l t u r e . MATERIALS AND METHODS: The two-rowed, spring b a r l e y c u l t i v a r Klages (Hordeum v u l g a t e L.) was used in t h i s study. Plants were grown as p r e v i o u s l y described (Marsolais and Kasha, 1985) and developing spikes were selected from the f i r s t t i l l e r s o f the p l a n t s . P r i o r to the removal and f i x a t i o n o f each o f the spikes in a c e t i c a c i d : alcohol ( 1 : 3 ) , the d i s t a n c e from the base o f
Offprint requests to: W. G. Wheatley
the f l a g l e a f to the penultimate l e a f was measured (cm) and recorded. A f t e r at l e a s t I day, the spikes were t r a n s f e r r e d to 70% alcohol and 6 to 9 anthers removed from 2 o r 3 f l o r e t s in the middle of the s p i k e . The anthers were stained according to the method of M i t c h e l l (1967) using d i n i t r o f l o r o b e n z e n e and Feulgen s t a i n s . A f t e r s t a i n i n g , the anthers were placed on microscope s l i d e s and the microspores removed by g e n t l e tapping with a d i s s e c t i n g needle. Permanent s l i d e s were made by using the method of Conger and F a i r c h i l d (1953). Measurements of c e l l areas, nuclear areas, maximum and minimum diameters of nuclei were made through a camera l u c i d a using a Zidas d i g i t i z i n g table~(Zeiss),.Nucl~a~ vqlumes wer~ c a l c u l a t e d accorm~ng to wnl~e anm uavlmson (Tg76). At l e a s t 50 c e l l s and nuclei were measured f o r each sample. The measurements of nuclear DNA content were made on 25 nuclei from each sample using a Zeiss MPV03 scanning microspectrophotometer at a wavelength o f 560 nm. RESULTS: Microspore nuclear size varied g r e a t l y w i t h i n anthers o f each sample and over the f i r s t m i t o t i c c e l l cycle of the microspore (Figures la and i b ) . Nuclei r e c e n t l y formed from the equational d i v i s i o n of meiosis ( i . e . e a r l y GI nuclei w i t h i n c e l l s forming t e t r a d s , f i g u r e 2a) had a mean nuclear volume o f 74.2 20.9 ~m3 ~ mean nuclear area = 22.7 + 4.0 pm2) compared to nuclei in prophase ( l a t e G2) o f the f i r s t p o l l e n m i t o s i s ( f i g u r e 2d) which had a mean nuclear volume of 402.3 + 130.2 ~m3 (mean nuclear area = 71.2 + 13.4 ~m2)~ On average, the nuclei increased in size by 5 . 4 - f o l d in terms of volume o r 3 . l - f o l d in terms of area over the course of the c e l l c y c l e . Even though there was c o n s i d e r a b l e v a r i a t i o n in e i t h e r nuclear volume or nuclear area, i t was apparent t h a t the c e l l c y c l e stage of the p o p u l a t i o n of microspores taken from anthers l o c a t e d at the middle o f a spike could be estimated from the mean nuclear area and/or mean nuclear volume (Figures la and I b ) . Anthers t h a t contained microspores having a mean nuclear #olume ranging from 104.1 + 35.6 to 157.9 + 58.8 ~ma (areas = 28.7 + 5.3 to 39.4 + 8.6 pm~) were in G1 phase as estimated by DNAmeasurements (Figure 2b). Anthers c o n t a i n i n g microspores having mean nuclear volume ranging from 246.6 + 93.0 to 312.2 + 130.2 pm3 (area = 51.6 + 10.4 t o 6 1 . O + 15.3 ~m~) had i n t e r m e d i a t e DNA values which Tndicated t h a t these p o p u l a t i o n s were undergoing DNA synthesis (S phase, f i g u r e 2c).
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MEAN NUCLF.AR DNA A M O U N T ( ~ . U . ) X Mltot]© p r o p h a l m
Figure 1: Nuclear size d i s t r i b u t i o n s and nuclear DNA amounts for microspores at various stages of the c e l l cycle. (Mean + S.D.) a) Nuclear area (pm2) versus nuclear DNA am-ount (A.U. = a r b i t r a r y u n i t s ) , b) Nuclear volume (lJm3) versus nuclear DNA amount.
Figure 2: Stages of microsporogenesls during f i r s t c e l l cycle in b a r l e y . a) Tetrad stage ( e a r l y G1, no microspore cell wall observed, small nucleus). b) Mid-uninucleate microspore in G1 ( c e l l s walls present, vacuole present and small nucleus displaced to periphery of microspore). c) Microspore in S phase ( s i m i l a r to 2b, nucleus slightly larger). d) Late-uninucleate microspore ( l a t e G2, large sperical nucleus in prophase of m i t o s i s ) . Bar denotes 10 lJm.
Tahle 1: Cell areas for c e l l s at stages during meiosis and microsporogenesis STAGE
CELL AREA (~m2) mean + S.D.
Microspores observed in the G2 phase had a mean nuclear volume of 336.4 + 118.4 j£m3 (area = 64.1 + 12.4 pm2): the mean DNA~ontent approached that the prophase n u c l e i . Part of the observed v a r i a t i o n in nuclear DNA content was due to b i r e f r i g e n c e of the microspore c e l l w a l l . In p a r t i c u l a r , t h i s contributed to the wider than expected range of mean DNA contents found in the GI microspores.
Meiotic prophase I Tetrad stage ( e a r l y G1) GI microspores G2 microspores (prophase of first mitosis)
A summary of the measurements of c e l l areas for c e l l s in prophase I of meiosis, t e t r a d stage, G1 microspores and microspores having nuclei undergoing prophase of the f i r s t pollen mitosis is presented in Table 1. Considerable v a r i a t i o n in c e l l size was observed over these stages. On average, the area of c e l l s at t e t r a d stage decreased to about 27% t h a t of c e l l s in meiotic prophase I. For microspores t h a t were in G1 of the c e l l cycle the mean c e l l area was about 5 to 6 - f o l d l a r g e r than the c e l l area of an average t e t r a d c e l l . Vacuole formation and expansion w i t h i n the developing microspores contributed g r e a t l y to t h i s increase in size. By the time of the f i r s t pollen mitosis the microspores size had increased by about 7 - f o l d compared to the t e t r a d c e l l s . Part of t h i s increase in size above the GI microspores appeared to be due to t h i c k e n i n g of the microspore cell wall.
The distance from the base of the f l a g l e a f blade to the base of the penultimate l e a f blade has been used as morphological t r a i t to assist in estimating the proper stages of the spike p r i o r to e x c i s i o n . Anthers which were removed from t i l l e r s in which the f l a g l e a f had emerged from 0 to 3 cm have proven to give the best percentage anther response f o l l o w i n g c u l t u r e (75-80% anther response, 10+15 c a l l i per responding a n t h e r ) . This observation applied to the c u l t i v a r Klages grown in our environment. These microspores were in G1 of the c e l l cylce and had comparatively small mean nuclear sizes and large vacuoles. These t r a i t s were c y t o l o g i c a l l y c h a r a c t e r i s t i c of the mid-uninucleate stage. Other observations i n d i c a t e d that spikes with anthers in which the f l a g l e a f had emerged beyond 3 cm could be
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340.5 + 91.4 T 481.4 T 632.1Z
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59,1 22,3 86,1 69.0
49 in GI, S or G2 o f the c e l l cycle and that the distance of flag l e a f emergence in which the f i r s t pollen mitosis was observed tended to be from 9.5 to 12 cm, although l a t e r and e a r l i e r stages of pollen formation were also found in t h i s range. Thus, while the use of t i l l e r in which the flag l e a f had recently emerged appeared to assist the selection of spikes which had anthers at the proper stage i t was not a s t r i c t c r i t e r i o n for selection of spikes which had mid-uninucleate (GI) microspores. DISCUSSION In t h i s study, the objective was to determine the precise c e l l cycle stage associated with high levels of anther response and p r o d u c t i v i t y . C e l l u l a r parameters such as c e l l area and nuclear size were measured and compared to spectrophotometric measurements of nuclear DNA content. Microspores within barley anthers increased considerably in c e l l size over the f i r s t c e l l cycle and was due p r i m a r i l y to vacuole formation and enlargement. By comparison, nuclear growth appeared more gradual over t h i s phase of development and exhibited wide v a r i a t i o n over the course of the c e l l cycle. Precise staging could not be accomplished by estimating the nuclear size of i n d i v i d u a l microspores. However, mean nuclear size estimates of microspores populations o f 50 c e l l s derived from individual anthers d i f f e r e n t i a t e d mid-uninucleate microspores in GI from those in G2 of the cell cycle and perhaps those in S phase. We suspect that imprecise staging of microspore development and v a r i a t i o n in microspore stage along the spike contributes s i g n i f i c a n t l y to sub-optimal levels of anther response and p r o d u c t i v i t y . In general, anthers taken from f l o r e t s in the middle o f the spike tended to be in more advanced stages of microsporogenesis compared to anthers in adjacent f l o r e t s . Anthers found in f l o r e t s in the bottom and top quarters of the spike lagged considerably behind in t h e i r development compared to anthers in the middle of the spike. Our results suport the suggestion by Sunderland eft a l . (1979) that mid-uninucleate and l a t e -
uninuclcu~u microsprores d i ~ e r ir~ huclear size. Howeven, the d i s t r i b u t i o n s of nuclear size of these stages overlapped due to inherent v a r i a b i l i t y and developmental asynchrony. Based on our measurements, the mid-uninucleate stage which gives high levels of anther response and p r o d u c t i v i t y corresponds to the G1 stage of the c e l l cycle. Although Sunderland et a l . (1979) indicated that the use of morphological-c h a r a c t e r i s t i c s of the t i l l e r s was not s a t i s f a c t o r y for d i s t i n g u i s h i n g between spikes bearing mid-uninucleate and late-uninucleate microspores, the distance of flag l e a f emergence proved to be a reasonably r e l i a b l e i n d i c a t o r of microspore stage with the c u l t i v a r Klages grown in our environment. Nevertheless, we recommend that spikes should be staged i n i t i a l l y using the r e l a t i v e l y simple method of estimating mean nuclear area. These results should be correlated with morphological t r a i t s such as flag l e a f emergence that vary with genotypes and environments. Improvements in anther staging procedures should lead to increased anther response and p r o d u c t i v i t y and a decrease in the error component within experir~ents. ACKNOWLEDGEMENTS Financial support provided by the Natural Sciences and Engineering Research Council of Canada and by the Ontario Ministry of Agriculture and Food is g r a t e f u l l y acknowledged. The technical assistance of Sylvia Flack in the operation of the scanning microspectrophotometer is greatly appreciated. REFERENCES: Conger, A.D., F a i r c h i l d , L.M. (1953) Stain Tech. 28: 281-283. Marsolais, A.A., Kasha, K.J. (1985) Can. J. Bot. (accepted) M i t c h e l l , J.P. (1967) J. Roy. Microsc. Soc. 87:106-123. Sun, C.S. (1978) In: Proc. Symp. Plant Tissue Culture. Peking, China, Science Press, Peking, China, pp. 117-125. Sunderland, N., Roberts, M., Evens, L.J., Wildon. D.C. (1979) J. Exp. Bot. 30:1133-1144 White, R.L., Davidson, D. (1976) Can. J. Genetic. Cytol. 18:385-396
