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Induction of typical cell division in isolated microspores of Lilium longiflorum and Tulipa gesneriana
Plant Science Letters, 17 (1980) 279--285 © Elsevier/North-HollandScientific Publishers Ltd.
279
INDUCTION OF TYPICAL CELL DIVISION IN ISOLATED MICROSPORES OF LILIUM L O N G I F L O R U M A N D TULIPA G E S N E R I A N A
ICHIRO T A N A K A and MICHIO ITO Department of Biology. Faculty of Science, Nagoya University,Nagoya 464 (Japan) (Received September 1st,1979) (Accepted November 15th, 1979)
SUMMARY
Techniques were successfully established for the culture of isolated microspores from lily and tulip anthers. The uninucleate microspores explanted at the G1 phase of the cell cycle were cultured through the mitotic cycle. Microspore division was polar and formed typical pollen grains showing a generative-within-vegetative cell arrangement. At maturity in the subsequent culture, the binucleated pollens germinated in a solid medium to produce a structure resembling a pollen tube.
INTRODUCTION The attempt to culture microsporocytes of higher plants for normal development leading to the formation of mature pollen is a novel approach to the study of the ontogeny and physiology of pollen development. Previous efforts to culture isolated microsporocytes through the meiotic cycle have been already successful [1], the method being useful for the solution of meiotic problems [ 2--4]. However, a method of microspore culture to achieve typical cell division resulting in the formation of two functionally different nuclei has not been previously described. A few studies have shown only partial success in anther cultures [5,6] which have been used for studies of microspore development [7,8], but such organ cultures still present a number of obstacles to physiological studies. It was for this reason that an attempt was made to induce normal development in explanted microspores which have the advantage of reduced interference by tapetal cells and of more efficient utilisation of substances such as isotopes and inhibitors in culture media [9]. In this paper we report on a procedure for the culture of isolated microspores from lily and tulip anthers.
280 MATERIALS AND METHODS
The sources of experimental materials were Lilium longiflorum c.v. Georgia, grown in a greenhouse and Tulipa gesneriana c.v. Mrs. Grullemans, stored in a cold r o o m at 8°C after the completion of meiosis which occurred naturally in early November. In the lily, the procedure used to identify successive stages in development of uninucleate microspores through G~--S--G: and M phases is based on the correlation b e t w e e n bud length and microspore development [9,10]. Under greenhouse conditions, the process o f microspore development begins at a bud length of 24 ram, and is completed approx. 15 days later, at a bud length of 58 mm (Fig. 1). This is followed by the development of binucleate pollen taking a further 10 days or so after microspore mitosis. Tulip bulbs subjected to low temperatures retained uninucleate microspores at the G~ phase for 4 months under continuous cold storage. Flower buds were surface-sterilised by immersion in 70% ethanol for 2 min and then dried with filter paper. The buds were aseptically opened and individual anthers were removed. Each anther was cut open at one end and the microspores were extruded by gentle squeezing from the end distal to the cut, except for lily buds longer than 49 mm in length. In the latter, microspores were collected from anthers with sharp forceps. Microspores from individual anthers were separately cultured as cell aggregates on filter paper placed on solid nutrient medium which consisted of mineral
100
I%)
F
50
24
30
40 GI
50 ~" S
60 (mm) 'IG2 MI
Fig. 1. Mitotic development of microspores of Lilium longiflorum in culture. The length of the flower bud (mm) corresponding to the different stages of the cell cycle at which the microspores were explanted, is marked on the abscissa. Ordinates represent the percentage of divided cells (e - ) and typical mitotic products (o o) to the total number of cultured microspores. These results were obtained after 2--10 days of culturing. The bars span all the frequencies found in several experiments.
281
salts and vitamins [1] supplemented with yeast extract (0.05%) and sucrose (17%), at pH 5.8. Difco-bacto agar was used to solidify the medium at concentrations of 0.8--4%. Cultures were grown in 7-cm petri dishes, each containing 10 ml of the medium. All cultures were maintained at 25°C, and were observed periodically. The survival of the cultures was expressed as the percentage of living cells of the total cell number. Viability was detected through an ordinary microscope by cytological criteria. Dead cells invariably showed shrunken nuclei which could be seen after staining with iron-acetic carmine. The nuclei in lily microspores were difficult to observe through usual procedures because of the existence of sculptured cell wall envelopes. For this reason, after carmine staining, occasional microspores were pressed down on a coverslip with sufficient force to extrude the cell content from their envelopes. The discrimination as to whether the development of microspores in the culture was typical or anomalous was based on cytological appearance and staining property. The medium for pollen germination was a 0.3 M sucrose solution solidified with agar at a concentration of 4% for the lily and 1% for the tulip, at pH 5.8. Germination tests were carried out by sowing cultured pollens on the germination medium at 25°C. RESULTS AND DISCUSSION
Lily microspores explanted from various lengths of buds ( 2 5 - 5 5 mm) survived well on the nutrient medium solidified with 1% agar, and showed no marked differences between stages of explantation. On the whole, 70--80% of the cells survived after the first day of culturing. The number of surviving cells decreased gradually with culture time, and after two weeks the survival rate was stationary at 40--50% of the cells. The nutritional conditions permitting survival of microspores in vitro appeared to be simple, but most of the living microspores showed accumulation of a large number of starch grains. Nuclear division in microspores was observed by the tenth day of culture and the binucleate microspores were produced at various frequencies, up to 3%. However, all showed cytological abnormalities, including binucleates with two, similarly stained, nuclei. Some modifications of nutrient composition, or incorporation of various growth regulators like kinetin [11] proved ineffective in attempting to augment normal development. Increased additions of agar to the nutrient medium induced typical microspore division to the extent as shown in Fig. 1. Figure 1 shows the frequencies of microspore division and of the production of typical pollen related at stages when the microspores were explanted on the solid medium containing 4% agar. It was difficult to determine precisely whether pollen so-produced was normal, but cytological appearance resembled that of the arrangement in normal generative-within-vegetativecells. The frequency of microspores from buds 45 mm or longer was approx. 70%; almost all divided ones were of a typical type. Thus, lily microspores may be explanted at
%,
(,
. .
': .-
•.
•
~,~--ii
:".~~L
.~_,e..~-' ,~ - . k -
~'~"
~-.~- ~
;..' . . ,
, ~,
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.
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•
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. . . . . . . .
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.,~,
Fig. 2. Cultured microspores of Lilium longiflorum (except F,H) and Tulipa gesnerlana (F,H). These microspores were explanted at the G I phase of the microspore cell cycle. (A) early prophase; (B) metaphase; (C) telophase; (D) a typical binucleated pollen; (E) an anomalous pollen with t w o nuclei equally stained. These cells, after 4 days of culture, were fixed, stained and extruded from their cell wall envelopes to be photographed. (F) tulip cells showing typical pollen configurations after 8 days of culture. (G,H) after 12 days of culture, cells developing a structure resembling a pollen tube on the germination medium. A--F, x 500; G, x 300; H, x 150.
~.
._
17 ~-
O0 O~
284
late G~ phase [9] and cultured through mitotic cycle giving rise to binucleate pollens. Relatively young microspores from buds shorter than 40 mm in length survived poorly on a hard medium such as solid agar, and no divisions were observed. Attempts to induce cell division in these microspores which are perhaps handicapped due to the very low level of metabolic activities [7,8] have so far failed, even when the cells were precultured on a soft solid medium (0.8% agar) followed by sequential additions of agar. Critical cytological investigations were undertaken with lily microspores isolated from 45-mm buds and cultured on the nutrient medium with 4% agar. At the initiation of the culture, a microspore is characterised by a sculptured wall and a large nucleus displaced from the cell's centre towards one side of the cell {Fig. 2A). After 3--4 days in the culture, the cells undergoing mitotic division were frequently observed (Fig. 2B, C). The division was a polar one, resulting in daughter cells of unequal size. The smaller of the two cells was attached to the wall with few cytoplasm or laid entirely within the cytoplasm of the larger cell. After division, the differences upon staining the two nuclei were clear; the nucleus in the smaller cell was densely stained with acetocarmine, whereas the larger was only lightly stained (Fig. 2D). No remarkable difference between cultured and intact materials was observed in mitosis or in subsequent development. Anomalous pollens with two equally-stained nuclei or produced by the equational cell division were occasionally observed (Fig. 2E). The rate of the mitotic cycle for intact microspores grown under greenhouse conditions compared to those grown in a culture was essentially the same. For example, microspores of 45-ram buds take approx. 4 days to complete mitosis in vivo [9], whereas in the culture the interval was approx. 3.5 days. The possibility of inducing typical cell division in isolated tulip microspores was successfully demonstrated when microspores were cultured in a nutrient medium containing agar {0.8%) and sucrose (17%). Invariably about 60% of these microspores underwent mitosis in the culture within 8 days and most binucleates showed typical pollen configurations {Fig. 2F). Such experiments could be repeated on bulbs maintained in cold storage for 4 months. The features of mitosis and the development of binucleates were quite similar to those in intact anthers; these processes occur naturally in vivo in the early spring. Development of the tulip microspores was not improved by raising the agar concentration as in lily. Thus, there are large differences between species in permissible agar concentrations, but in respective species the microspore environment in vitro is observed to be similar to that in vivo with respect to the moist condition [9]. Attempts to initiate pollen tube development in pollens cultured from the uninucleate microspore stage (GI phase) have been made. The pollens, in which mitosis had already completed followed by subsequent culturing in the same medium for several days, were observed to develop a protuberance in the germination medium. Although such occurrences were relatively
285 i n f r e q u e n t ( 2 0 - - 4 0 % f o r t h e lily, 2 0 - - 3 0 % f o r t h e tulip), t h e p r o t u b e r a n c e o n t h e p o l l e n c o n t i n u e d t o e l o n g a t e and f o r m e d a s t r u c t u r e resembling a pollen t u b e {Fig. 2G, H). Dividing nuclei o r t h r e e nuclei w e r e occasionally o b s e r v e d in t h e s e s t r u c t u r e s ; this is a n a l o g o u s t o p h e n o m e n a which o c c u r at t h e final step o f t h e o n t o g e n y o f p o l l e n d e v e l o p m e n t . We have n o t t e s t e d g e r m i n a t i o n o f c u l t u r e d pollens o n stigma, b u t t h e a b o v e results p o i n t t o t h e possibility o f in v i t r o c u l t u r e for t h e f o r m a t i o n o f m a t u r e pollens f r o m t h e y o u n g m i c m s p o r e stage t h r o u g h t w o cell cycles. REFERENCES
1 2 3 4 5 6 7 8 9 10 11
M. Ito and H. Stem, Dev. Biol., 16 (1967) 36. M. Ito, Y. Hotta and H. Stem, Dev. Biol., 16 (1967) 54. H. Stern and Y. Hotta, Annu. Rev. Genet., 7 (1974) 37. N. Kurata and M. Ito, Cell Stmct. Funct., 3 (1978) 349. A.H. Sparrow, V. Pond and S. Kojan, Am. J. Bot., 42 (1955) 384. N. Sunderland and F.M. Wicks, J. Exp. Bot., 22 (1971) 213. J.H. Taylor and R.D. McMaster, Chromosoma, 6 (1954) 489. Y. Hotta and H. Stem, J. Cell Biol., 25 (1965) 99. I. Tanaka, T. Taguchi and M. Ito, Bot. Mag. Tokyo, 92 (1979) 291. R.O. Erickson, Am. J. Bot., 35 (1948) 729. G.W.R. Walker and J.F. Dietrich, Can. J. Genet. Cytol., 3 (1961) 170.
279
INDUCTION OF TYPICAL CELL DIVISION IN ISOLATED MICROSPORES OF LILIUM L O N G I F L O R U M A N D TULIPA G E S N E R I A N A
ICHIRO T A N A K A and MICHIO ITO Department of Biology. Faculty of Science, Nagoya University,Nagoya 464 (Japan) (Received September 1st,1979) (Accepted November 15th, 1979)
SUMMARY
Techniques were successfully established for the culture of isolated microspores from lily and tulip anthers. The uninucleate microspores explanted at the G1 phase of the cell cycle were cultured through the mitotic cycle. Microspore division was polar and formed typical pollen grains showing a generative-within-vegetative cell arrangement. At maturity in the subsequent culture, the binucleated pollens germinated in a solid medium to produce a structure resembling a pollen tube.
INTRODUCTION The attempt to culture microsporocytes of higher plants for normal development leading to the formation of mature pollen is a novel approach to the study of the ontogeny and physiology of pollen development. Previous efforts to culture isolated microsporocytes through the meiotic cycle have been already successful [1], the method being useful for the solution of meiotic problems [ 2--4]. However, a method of microspore culture to achieve typical cell division resulting in the formation of two functionally different nuclei has not been previously described. A few studies have shown only partial success in anther cultures [5,6] which have been used for studies of microspore development [7,8], but such organ cultures still present a number of obstacles to physiological studies. It was for this reason that an attempt was made to induce normal development in explanted microspores which have the advantage of reduced interference by tapetal cells and of more efficient utilisation of substances such as isotopes and inhibitors in culture media [9]. In this paper we report on a procedure for the culture of isolated microspores from lily and tulip anthers.
280 MATERIALS AND METHODS
The sources of experimental materials were Lilium longiflorum c.v. Georgia, grown in a greenhouse and Tulipa gesneriana c.v. Mrs. Grullemans, stored in a cold r o o m at 8°C after the completion of meiosis which occurred naturally in early November. In the lily, the procedure used to identify successive stages in development of uninucleate microspores through G~--S--G: and M phases is based on the correlation b e t w e e n bud length and microspore development [9,10]. Under greenhouse conditions, the process o f microspore development begins at a bud length of 24 ram, and is completed approx. 15 days later, at a bud length of 58 mm (Fig. 1). This is followed by the development of binucleate pollen taking a further 10 days or so after microspore mitosis. Tulip bulbs subjected to low temperatures retained uninucleate microspores at the G~ phase for 4 months under continuous cold storage. Flower buds were surface-sterilised by immersion in 70% ethanol for 2 min and then dried with filter paper. The buds were aseptically opened and individual anthers were removed. Each anther was cut open at one end and the microspores were extruded by gentle squeezing from the end distal to the cut, except for lily buds longer than 49 mm in length. In the latter, microspores were collected from anthers with sharp forceps. Microspores from individual anthers were separately cultured as cell aggregates on filter paper placed on solid nutrient medium which consisted of mineral
100
I%)
F
50
24
30
40 GI
50 ~" S
60 (mm) 'IG2 MI
Fig. 1. Mitotic development of microspores of Lilium longiflorum in culture. The length of the flower bud (mm) corresponding to the different stages of the cell cycle at which the microspores were explanted, is marked on the abscissa. Ordinates represent the percentage of divided cells (e - ) and typical mitotic products (o o) to the total number of cultured microspores. These results were obtained after 2--10 days of culturing. The bars span all the frequencies found in several experiments.
281
salts and vitamins [1] supplemented with yeast extract (0.05%) and sucrose (17%), at pH 5.8. Difco-bacto agar was used to solidify the medium at concentrations of 0.8--4%. Cultures were grown in 7-cm petri dishes, each containing 10 ml of the medium. All cultures were maintained at 25°C, and were observed periodically. The survival of the cultures was expressed as the percentage of living cells of the total cell number. Viability was detected through an ordinary microscope by cytological criteria. Dead cells invariably showed shrunken nuclei which could be seen after staining with iron-acetic carmine. The nuclei in lily microspores were difficult to observe through usual procedures because of the existence of sculptured cell wall envelopes. For this reason, after carmine staining, occasional microspores were pressed down on a coverslip with sufficient force to extrude the cell content from their envelopes. The discrimination as to whether the development of microspores in the culture was typical or anomalous was based on cytological appearance and staining property. The medium for pollen germination was a 0.3 M sucrose solution solidified with agar at a concentration of 4% for the lily and 1% for the tulip, at pH 5.8. Germination tests were carried out by sowing cultured pollens on the germination medium at 25°C. RESULTS AND DISCUSSION
Lily microspores explanted from various lengths of buds ( 2 5 - 5 5 mm) survived well on the nutrient medium solidified with 1% agar, and showed no marked differences between stages of explantation. On the whole, 70--80% of the cells survived after the first day of culturing. The number of surviving cells decreased gradually with culture time, and after two weeks the survival rate was stationary at 40--50% of the cells. The nutritional conditions permitting survival of microspores in vitro appeared to be simple, but most of the living microspores showed accumulation of a large number of starch grains. Nuclear division in microspores was observed by the tenth day of culture and the binucleate microspores were produced at various frequencies, up to 3%. However, all showed cytological abnormalities, including binucleates with two, similarly stained, nuclei. Some modifications of nutrient composition, or incorporation of various growth regulators like kinetin [11] proved ineffective in attempting to augment normal development. Increased additions of agar to the nutrient medium induced typical microspore division to the extent as shown in Fig. 1. Figure 1 shows the frequencies of microspore division and of the production of typical pollen related at stages when the microspores were explanted on the solid medium containing 4% agar. It was difficult to determine precisely whether pollen so-produced was normal, but cytological appearance resembled that of the arrangement in normal generative-within-vegetativecells. The frequency of microspores from buds 45 mm or longer was approx. 70%; almost all divided ones were of a typical type. Thus, lily microspores may be explanted at
%,
(,
. .
': .-
•.
•
~,~--ii
:".~~L
.~_,e..~-' ,~ - . k -
~'~"
~-.~- ~
;..' . . ,
, ~,
" ~
O0
.
•
,....
•
.
?'i.
, ~ . . . .
"
. . ~' . ~ . i . ~ 2 ~
..
.
. . . . . . . .
•
d
• 5
".~
•
•
~
' ~
' ~11
•
,
.~!
.,~,
Fig. 2. Cultured microspores of Lilium longiflorum (except F,H) and Tulipa gesnerlana (F,H). These microspores were explanted at the G I phase of the microspore cell cycle. (A) early prophase; (B) metaphase; (C) telophase; (D) a typical binucleated pollen; (E) an anomalous pollen with t w o nuclei equally stained. These cells, after 4 days of culture, were fixed, stained and extruded from their cell wall envelopes to be photographed. (F) tulip cells showing typical pollen configurations after 8 days of culture. (G,H) after 12 days of culture, cells developing a structure resembling a pollen tube on the germination medium. A--F, x 500; G, x 300; H, x 150.
~.
._
17 ~-
O0 O~
284
late G~ phase [9] and cultured through mitotic cycle giving rise to binucleate pollens. Relatively young microspores from buds shorter than 40 mm in length survived poorly on a hard medium such as solid agar, and no divisions were observed. Attempts to induce cell division in these microspores which are perhaps handicapped due to the very low level of metabolic activities [7,8] have so far failed, even when the cells were precultured on a soft solid medium (0.8% agar) followed by sequential additions of agar. Critical cytological investigations were undertaken with lily microspores isolated from 45-mm buds and cultured on the nutrient medium with 4% agar. At the initiation of the culture, a microspore is characterised by a sculptured wall and a large nucleus displaced from the cell's centre towards one side of the cell {Fig. 2A). After 3--4 days in the culture, the cells undergoing mitotic division were frequently observed (Fig. 2B, C). The division was a polar one, resulting in daughter cells of unequal size. The smaller of the two cells was attached to the wall with few cytoplasm or laid entirely within the cytoplasm of the larger cell. After division, the differences upon staining the two nuclei were clear; the nucleus in the smaller cell was densely stained with acetocarmine, whereas the larger was only lightly stained (Fig. 2D). No remarkable difference between cultured and intact materials was observed in mitosis or in subsequent development. Anomalous pollens with two equally-stained nuclei or produced by the equational cell division were occasionally observed (Fig. 2E). The rate of the mitotic cycle for intact microspores grown under greenhouse conditions compared to those grown in a culture was essentially the same. For example, microspores of 45-ram buds take approx. 4 days to complete mitosis in vivo [9], whereas in the culture the interval was approx. 3.5 days. The possibility of inducing typical cell division in isolated tulip microspores was successfully demonstrated when microspores were cultured in a nutrient medium containing agar {0.8%) and sucrose (17%). Invariably about 60% of these microspores underwent mitosis in the culture within 8 days and most binucleates showed typical pollen configurations {Fig. 2F). Such experiments could be repeated on bulbs maintained in cold storage for 4 months. The features of mitosis and the development of binucleates were quite similar to those in intact anthers; these processes occur naturally in vivo in the early spring. Development of the tulip microspores was not improved by raising the agar concentration as in lily. Thus, there are large differences between species in permissible agar concentrations, but in respective species the microspore environment in vitro is observed to be similar to that in vivo with respect to the moist condition [9]. Attempts to initiate pollen tube development in pollens cultured from the uninucleate microspore stage (GI phase) have been made. The pollens, in which mitosis had already completed followed by subsequent culturing in the same medium for several days, were observed to develop a protuberance in the germination medium. Although such occurrences were relatively
285 i n f r e q u e n t ( 2 0 - - 4 0 % f o r t h e lily, 2 0 - - 3 0 % f o r t h e tulip), t h e p r o t u b e r a n c e o n t h e p o l l e n c o n t i n u e d t o e l o n g a t e and f o r m e d a s t r u c t u r e resembling a pollen t u b e {Fig. 2G, H). Dividing nuclei o r t h r e e nuclei w e r e occasionally o b s e r v e d in t h e s e s t r u c t u r e s ; this is a n a l o g o u s t o p h e n o m e n a which o c c u r at t h e final step o f t h e o n t o g e n y o f p o l l e n d e v e l o p m e n t . We have n o t t e s t e d g e r m i n a t i o n o f c u l t u r e d pollens o n stigma, b u t t h e a b o v e results p o i n t t o t h e possibility o f in v i t r o c u l t u r e for t h e f o r m a t i o n o f m a t u r e pollens f r o m t h e y o u n g m i c m s p o r e stage t h r o u g h t w o cell cycles. REFERENCES
1 2 3 4 5 6 7 8 9 10 11
M. Ito and H. Stem, Dev. Biol., 16 (1967) 36. M. Ito, Y. Hotta and H. Stem, Dev. Biol., 16 (1967) 54. H. Stern and Y. Hotta, Annu. Rev. Genet., 7 (1974) 37. N. Kurata and M. Ito, Cell Stmct. Funct., 3 (1978) 349. A.H. Sparrow, V. Pond and S. Kojan, Am. J. Bot., 42 (1955) 384. N. Sunderland and F.M. Wicks, J. Exp. Bot., 22 (1971) 213. J.H. Taylor and R.D. McMaster, Chromosoma, 6 (1954) 489. Y. Hotta and H. Stem, J. Cell Biol., 25 (1965) 99. I. Tanaka, T. Taguchi and M. Ito, Bot. Mag. Tokyo, 92 (1979) 291. R.O. Erickson, Am. J. Bot., 35 (1948) 729. G.W.R. Walker and J.F. Dietrich, Can. J. Genet. Cytol., 3 (1961) 170.