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Histological observations on the initiation of the vegetative apex in tulip seeds cultured under low temperatures
Scientia Horticulturae, 13 (1980) 161--171
161
Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
HISTOLOGICAL OBSERVATIONS ON THE INITIATION OF THE V E G E T A T I V E APEX IN T U L I P SEEDS C U L T U R E D U N D E R LOW TEMPERATURES
YOSHIJI NIIMI
Faculty of Agriculture, Horticultural Laboratory, Niigata University, Niigata, 950-21 (Japan) Present temporary address: Dept. Botany, University of Nijmegen, Tournooiveld, Nijmegen, The Netherlands. (Accepted for publication 12 December 1979)
ABSTRACT Niimi, Y., 1980. Histological observations on the initiation of the vegetative apex in tulip seeds cultured under low temperatures. Scientia Hortic., 13: 161--171. The tulip embryo grows upward within the seed under low temperature at first and becomes nearly the same length as the seed after about 40 days of inoculation on an inorganic salt medium, but the distinct cell divisions of the undifferentiated vegetative apex can scarcely be ascertained during this period. After germination, the seedlings grow downward and the real divisions of the apex begin: at first, anticlinal or periclinal divisions take place actively in the deeper layers of the apex, followed by frequent anticlinal divisions in the peripheral layers as well as in the deeper layers. As a result, the vegetative apex can be detected distinctly within 70 days of inoculation as a mass of cells which becomes a slightly domed apex.
INTRODUCTION In a previous p a p e r t h e a u t h o r s h o w e d t h a t l o w t e m p e r a t u r e s are necessary n o t o n l y f o r t h e e l o n g a t i o n o f t h e c o t y l e d o n or t h e b r e a k i n g o f d o r m a n cy, b u t also f o r t h e initiation o f t h e b u l b p r i m o r d i u m in the tulip seed (Niimi, 1 9 7 8 ) . T h e latter f a c t suggests t h a t t h e tulip seed p r o b a b l y c o n t a i n s an i m m a t u r e e m b r y o a n d t h a t certain changes o c c u r in t h e vegetative a p e x d u r i n g l o w - t e m p e r a t u r e t r e a t m e n t . H o w e v e r , these possibilities were n o t exa m i n e d in detail. T h e p r e s e n t s t u d y was carried o u t t o clarify m o r p h o l o g i c a l l y t h e developm e n t o f t h e vegetative a p e x u n d e r l o w t e m p e r a t u r e . MATERIALS AND METHODS
Seeds o f Tulipa gesneriana 'William P i t t ' × ' D u k e o f W e l l i n g t o n ' were
162
prepared in the same way as described in the previous paper (Niimi, 1978). After sterilization in 10% calcium hypochlorite solution for a b o u t 20 min, the seeds were rinsed 3 times in sterile distilled water. Each seed was placed in a test-tube (18 × 180 mm) containing Nitsch's inorganic medium {1951), solidified with 0.7% agar. The medium was adjusted to pH 5.0 with 0.1 N HC1 and 0.1 N NaOH before the addition of agar. Inoculated test-tubes were kept at 4 + I ° C for 70 days. Some of them, as control, were kept in a r o o m at 20 + I°C. Embryos, before germination, were excised every 10 days from the seeds with tweezers, transversely cut in half and then the lower parts were fixed. After germination, relatively larger portions, ranging from 5 to 10 mm long including the vegetative apex, were fixed 46, 60 and 70 days after inoculation. The excised tissues were fixed in a mixture of 3% glutaraldehyde and 4% acrolein in 0.025 M phosphate buffer pH 6.8 for 3 h at a b o u t 4°C (Gaff et al., 1976). They were rinsed several times in a cold phosphate buffer and dehydrated in an alcohol--propylene oxide series, and then embedded in the standard resin of Spurt (1969). The m o u n t e d specimens were transversely or longitudinally cut a b o u t 1.5 p m thick with a glass knife on a Jeol J u m 7 ultramicrotome, and the sections were floated on a drop of distilled water which was later evaporated on a h o t plate to adhere the sections to the microscope slide. The sections were stained according to the method of Feder and O'Brien (1968) with a slight modification; the periodic-acid-Shift's (PAS) reaction, coupled with s-amylase extraction, was used to detect insoluble carbohydrates and the sections were counter-stained with 0.05% toludine blue-O in 0.02 M benzoate buffer, pH 4.4 (Sidman et al., 1961) for 10--15 min at a b o u t 60 ° C. OBSERVATIONS E x t e r n a l observations. -- Figure 1 shows the growth of embryos at 4 ° C.
Each point represents the mean o f 15--20 seeds. A tulip embryo, as compared with a seed of a b o u t 10 m m long, is a b o u t 3 mm long at inoculation (Fig. 1A). The e m b r y o scarcely grows during 20 days. Thereafter, it gradually begins to elongate upward within the seed and becomes nearly the same length as the seed a b o u t 40 days after inoculation (Fig. 1B), and more than 50% of the seeds germinate after 46 days from inoculation (Fig. 1C). When the radicle becomes 1--2 mm long, it begins to dip down into the agar (Fig. 1D). Sixty to 70 days after inoculation, the c o t y l e d o n and radicle can be distinguished from each other b y the naked eye because of their active growth (Fig. 1E), b u t the cotyledon never appears above the agar under low temperature before the seedling is cultured under warm conditions. M o r p h o l o g i c a l o b s e r v a t i o n s o n the d e v e l o p m e n t o f the vegetative a p e x u n d e r l o w - t e m p e r a t u r e conditions. -- The e m b r y o of an untreated seed has the fol-
163 30
~,20 A
¢, 10 ¢¢ ¢D
0
10 20 30 40 46 6"0 70 Ouration of,days treated at 4°C Fig.1. Changes in total length of embryo or cotyledon at 4 ° C.A, untreated seed;B, 40day-old seed;C, 42-day-old seed;D, 46-day-old seed;E, 70-day-old seed.
lowing t y p i c a l c o n s t r u c t i o n : o n e o f t h e c o t y l e d o n a r y p r o c a m b i a l strands is linked t o t h e h y p o c o t y l and t h e o t h e r is c o n n e c t e d w i t h t h e vegetative a p e x ; t h e u n d i f f e r e n t i a t e d vegetative a p e x is easily distinguished f r o m the c o t y l e d o n a n d h y p o c o t y l tissues b y its lightly stained cells o f various sizes (Figs. 2 and 3).
Fig. 2. Longitudinal section of untreated embryo. In all figures the marker (bottom left) represents 50 pro. ps = procambial strands; v = vegetative apex.
164
Fig. 3. Transverse s e c t i o n o f u n t r e a t e d e m b r y o , ps = p r o c a m b i a l s t r a n d s ; v = vegetative apex.
Histological changes are not distinguishable in the embryos of the seeds after 5 and 10 days of incubation, but in the 10-day-old embryo, insoluble carbohydrates are observed in all cells except those of the vegetative apex and procambial strands (Fig. 4). The appearance of insoluble carbohydrates suggests that certain responses take place in the embryo during those 10 days.
Fig. 4. L o n g i t u d i n a l s e c t i o n o f a 10-day-old e m b r y o s h o w i n g t h e first a p p e a r a n c e o f insoluble c a r b o h y d r a t e s , e = e p i d e r m i s ; v = vegetative apex.
165
The first morphological changes of the whole embryo are observed 20 days after inoculation. Although the cells of the vegetative apex still seem to be mitotically quiescent, procambial strands, as compared with those of the untreated seed in Fig. 2, are more evident (Fig. 5). In addition, waved cell walls occur, suggesting that the divisions of cells of the vegetative apex may begin soon (Fig. 6).
Fig. 5. Longitudinal section of a 2 0 - d a y - o l d embryo showing that procambial strands become more evident, as compared with the control in Fig. 2. p s = procambial strands; v = vegetative a p e x .
Fig. 6. Transverse section of a 2 0 - d a y - o l d embryo showing the waved cell walls of vegetative apex cells, v = v e g e t a t i v e a p e x .
166
Certain cells of the vegetative apex begin to divide 30 days after inoculation; their divisions are particularly observed in the cells which adjoin procambial strands and also in those of the deeper layers (Fig. 7). It is in the germinated seed that the real cell divisions of the vegetative apex start, and
Fig. 7. L o n g i t u d i n a l s e c t i o n o f a 3 0 - d a y - o l d e m b r y o s h o w i n g t h e first cell divisions o f t h e vegetative apex. e = e p i d e r m i s ; v = vegetative apex.
Fig. 8. L o n g i t u d i n a l s e c t i o n o f a 4 6 - d a y - o l d e m b r y o j u s t a f t e r g e r m i n a t i o n , s h o w i n g t h e real cell divisions o f t h e vegetative a p e x a n d t h e m o r e e v i d e n t p r o c a m b i a l strands, ps = p r o c a m b i a l s t r a n d s ; v = vegetative apex.
167 the first p r o t o x y l e m elements appear in their procambial strands (Figs. 8 and 9). During a period of 30--46 days, the anticlinal and periclinal cell divisions take place in the deeper layers of the apicies much more predominantly than the anticlinal divisions in the peripheral layers, b u t cell divisions are scarcely detectable in the embryos of seed cultured at 20°C for 40 days (Fig. 10).
Fig. 9. Transverse section of a 46-day-old embryo showing the cell divisions of the vegetative apex and the first protoxylem, px = protoxylem; v = vegetative apex.
After 60 days from inoculation, the anticlinal divisions in the peripheral layers of the vegetative apex, as well as the anticlinal or periclinal divisions in the deeper layers, occur more frequently and the mass of cells can distinctly be detected as a vegetative apex, b u t it is still flat (Fig. 11). The vegetative apex continuously increases in volume by both anticlinal and periclinal cell divisions and becomes a slightly d o m e d apex consisting of cells with dense cytoplasm and large nuclei, although procambial strands relating to the initiation of a leaf primordium are n o t observable in the apex at this stage (Figs. 12 and 13). On the other hand, such cell divisions and histological changes of the vegetative apex are scarcely observed in the embryos of seed cultured at 20°C for 70 days (Fig. 14).
168
Fig. 10. Longitudinal section of a 40-day-old embryo cultured at 20°C, showing the very limited extent of cell divisions of the vegetative apex and a small quantity of insoluble carbohydrates, ps = procambial strands; v = vegetative apex.
Fig. 11. Longitudinal section of a 60-day-old seedling showing the anticlinal or periclinal divisions in the deeper layers of a nearly fiat vegetative apex. e = epidermis; px = protoxylem; v = vegetative apex.
169
!i,f+~.~
Im
i0
f-
Fig. 12. Longitudinal s e c t i o n o f a 7 0 - d a y - o l d seedling s h o w i n g a slightly d o m e d a p e x consisting o f cells w i t h dense c y t o p l a s m and large nuclei.
Fig. 13. Transverse s e c t i o n o f a 7 0 - d a y - o l d seedling s h o w i n g an a l m o s t circular vegetative a p e x and vascular bundles, p x = p r o t o x y l e m ; v = vegetative a p e x ; vb = vascular bundles.
170
Fig. 14. Longitudinal section of a 70-day-old embryo cultured at 20°C, showing the very extent of cell divisions o f a vegetative apex. e = e p i d e r m i s ; v ffi v e g e t a t i v e a p e x .
limited
DISCUSSION L o w temperatures for the germination of tulip seeds in vivo are required for a b o u t 40--50 days (Sisa and Higuchi, 1967). The present results show that the tulip e m b r y o elongates within the seed at first and its radicle starts to emerge 40 days after inoculation. Thus, the low-temperature requirement for germination of the seeds of Tulipa gesneriana seems to be satisfied in 40--50 days. When germinated seeds with 1 to 2-mm long radicles are transferred to warm conditions at 20°C (Fig. 1C), all elongate their cotyledons upward, b u t only t w o fifths of these seedlings form bulbs (unpublished data). This result agrees fairly closely with that of a previous paper (Niimi, 1978). Thus, it seems that the d o r m a n c y of the tulip seed is ended as soon as the radicle appears o u t of the seed coat, b u t a longer duration of low temperature is necessary for the undifferentiated vegetative apex to develop fully. This point ~s also clarified by the present histological observations on the
171
development of the vegetative apex; the real cell divisions of the undifferentiated vegetative apex activated by low temperatures begin with the seed germination and the flat apex develops into a slightly domed one by 70 days from inoculation. These results also show that a relatively longer period of low temperatures is required for the full development of the vegetative apex. CONCLUSION
When the tulip seed is naturally dispersed from the capsule, the embryo is still immature because the vegetative apex is not fully developed. The cells of the undifferentiated apex are activated by low temperatures and the real divisions begin after seed germination. Thus, low temperatures affect the further differentiation of the vegetative apex as well as the induction of seed germination. ACKNOWLEDGEMENTS
The author is grateful to the Emeritus Professor Dr. M. Kumazawa of Nagoya University and to Professor Dr. T. Takano of Meijo University for valuable discussion and advice.
REFERENCES Feder, N. and O'Brien, T.P., 1968. Plant microtechnique: some principles and new methods. Am. J. Bot., 55: 123--142. Gaff, D.E., Zee, S.-Y. and O'Brien, T.P., 1976. The fine structure of dehydrated and reviving leaves of Borya nitida Lalill. -- a desiccation tolerant plant. Aust. J. Bot., 24: 225--236. Niimi, Y., 1978. Influence of low and high temperatures on the initiation and the development o f a bulb primordium in isolated tulip embryos. Scientia Hortic., 9: 61--69. Nitsch, J.P., 1951. Growth and development in vitro of excised ovaries. Am. J. Bot., 38: 566--577. Sidman, R.L., Mottla, P.A. and Feder, N., 1961. Improved polyester wax embedding for histology. Stain Technol., 36: 279--284. Sisa, M. and Higuchi, H., 1967. Studies on shortening the juvenile phase of tulips under controlled environments. I. On the germination of seed and growth of seedling. Jpn. J. Breed., 17: 122--130. (In Japanese with English summary.) Spurt, A.R., 1969. A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res., 26: 31--43.
161
Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
HISTOLOGICAL OBSERVATIONS ON THE INITIATION OF THE V E G E T A T I V E APEX IN T U L I P SEEDS C U L T U R E D U N D E R LOW TEMPERATURES
YOSHIJI NIIMI
Faculty of Agriculture, Horticultural Laboratory, Niigata University, Niigata, 950-21 (Japan) Present temporary address: Dept. Botany, University of Nijmegen, Tournooiveld, Nijmegen, The Netherlands. (Accepted for publication 12 December 1979)
ABSTRACT Niimi, Y., 1980. Histological observations on the initiation of the vegetative apex in tulip seeds cultured under low temperatures. Scientia Hortic., 13: 161--171. The tulip embryo grows upward within the seed under low temperature at first and becomes nearly the same length as the seed after about 40 days of inoculation on an inorganic salt medium, but the distinct cell divisions of the undifferentiated vegetative apex can scarcely be ascertained during this period. After germination, the seedlings grow downward and the real divisions of the apex begin: at first, anticlinal or periclinal divisions take place actively in the deeper layers of the apex, followed by frequent anticlinal divisions in the peripheral layers as well as in the deeper layers. As a result, the vegetative apex can be detected distinctly within 70 days of inoculation as a mass of cells which becomes a slightly domed apex.
INTRODUCTION In a previous p a p e r t h e a u t h o r s h o w e d t h a t l o w t e m p e r a t u r e s are necessary n o t o n l y f o r t h e e l o n g a t i o n o f t h e c o t y l e d o n or t h e b r e a k i n g o f d o r m a n cy, b u t also f o r t h e initiation o f t h e b u l b p r i m o r d i u m in the tulip seed (Niimi, 1 9 7 8 ) . T h e latter f a c t suggests t h a t t h e tulip seed p r o b a b l y c o n t a i n s an i m m a t u r e e m b r y o a n d t h a t certain changes o c c u r in t h e vegetative a p e x d u r i n g l o w - t e m p e r a t u r e t r e a t m e n t . H o w e v e r , these possibilities were n o t exa m i n e d in detail. T h e p r e s e n t s t u d y was carried o u t t o clarify m o r p h o l o g i c a l l y t h e developm e n t o f t h e vegetative a p e x u n d e r l o w t e m p e r a t u r e . MATERIALS AND METHODS
Seeds o f Tulipa gesneriana 'William P i t t ' × ' D u k e o f W e l l i n g t o n ' were
162
prepared in the same way as described in the previous paper (Niimi, 1978). After sterilization in 10% calcium hypochlorite solution for a b o u t 20 min, the seeds were rinsed 3 times in sterile distilled water. Each seed was placed in a test-tube (18 × 180 mm) containing Nitsch's inorganic medium {1951), solidified with 0.7% agar. The medium was adjusted to pH 5.0 with 0.1 N HC1 and 0.1 N NaOH before the addition of agar. Inoculated test-tubes were kept at 4 + I ° C for 70 days. Some of them, as control, were kept in a r o o m at 20 + I°C. Embryos, before germination, were excised every 10 days from the seeds with tweezers, transversely cut in half and then the lower parts were fixed. After germination, relatively larger portions, ranging from 5 to 10 mm long including the vegetative apex, were fixed 46, 60 and 70 days after inoculation. The excised tissues were fixed in a mixture of 3% glutaraldehyde and 4% acrolein in 0.025 M phosphate buffer pH 6.8 for 3 h at a b o u t 4°C (Gaff et al., 1976). They were rinsed several times in a cold phosphate buffer and dehydrated in an alcohol--propylene oxide series, and then embedded in the standard resin of Spurt (1969). The m o u n t e d specimens were transversely or longitudinally cut a b o u t 1.5 p m thick with a glass knife on a Jeol J u m 7 ultramicrotome, and the sections were floated on a drop of distilled water which was later evaporated on a h o t plate to adhere the sections to the microscope slide. The sections were stained according to the method of Feder and O'Brien (1968) with a slight modification; the periodic-acid-Shift's (PAS) reaction, coupled with s-amylase extraction, was used to detect insoluble carbohydrates and the sections were counter-stained with 0.05% toludine blue-O in 0.02 M benzoate buffer, pH 4.4 (Sidman et al., 1961) for 10--15 min at a b o u t 60 ° C. OBSERVATIONS E x t e r n a l observations. -- Figure 1 shows the growth of embryos at 4 ° C.
Each point represents the mean o f 15--20 seeds. A tulip embryo, as compared with a seed of a b o u t 10 m m long, is a b o u t 3 mm long at inoculation (Fig. 1A). The e m b r y o scarcely grows during 20 days. Thereafter, it gradually begins to elongate upward within the seed and becomes nearly the same length as the seed a b o u t 40 days after inoculation (Fig. 1B), and more than 50% of the seeds germinate after 46 days from inoculation (Fig. 1C). When the radicle becomes 1--2 mm long, it begins to dip down into the agar (Fig. 1D). Sixty to 70 days after inoculation, the c o t y l e d o n and radicle can be distinguished from each other b y the naked eye because of their active growth (Fig. 1E), b u t the cotyledon never appears above the agar under low temperature before the seedling is cultured under warm conditions. M o r p h o l o g i c a l o b s e r v a t i o n s o n the d e v e l o p m e n t o f the vegetative a p e x u n d e r l o w - t e m p e r a t u r e conditions. -- The e m b r y o of an untreated seed has the fol-
163 30
~,20 A
¢, 10 ¢¢ ¢D
0
10 20 30 40 46 6"0 70 Ouration of,days treated at 4°C Fig.1. Changes in total length of embryo or cotyledon at 4 ° C.A, untreated seed;B, 40day-old seed;C, 42-day-old seed;D, 46-day-old seed;E, 70-day-old seed.
lowing t y p i c a l c o n s t r u c t i o n : o n e o f t h e c o t y l e d o n a r y p r o c a m b i a l strands is linked t o t h e h y p o c o t y l and t h e o t h e r is c o n n e c t e d w i t h t h e vegetative a p e x ; t h e u n d i f f e r e n t i a t e d vegetative a p e x is easily distinguished f r o m the c o t y l e d o n a n d h y p o c o t y l tissues b y its lightly stained cells o f various sizes (Figs. 2 and 3).
Fig. 2. Longitudinal section of untreated embryo. In all figures the marker (bottom left) represents 50 pro. ps = procambial strands; v = vegetative apex.
164
Fig. 3. Transverse s e c t i o n o f u n t r e a t e d e m b r y o , ps = p r o c a m b i a l s t r a n d s ; v = vegetative apex.
Histological changes are not distinguishable in the embryos of the seeds after 5 and 10 days of incubation, but in the 10-day-old embryo, insoluble carbohydrates are observed in all cells except those of the vegetative apex and procambial strands (Fig. 4). The appearance of insoluble carbohydrates suggests that certain responses take place in the embryo during those 10 days.
Fig. 4. L o n g i t u d i n a l s e c t i o n o f a 10-day-old e m b r y o s h o w i n g t h e first a p p e a r a n c e o f insoluble c a r b o h y d r a t e s , e = e p i d e r m i s ; v = vegetative apex.
165
The first morphological changes of the whole embryo are observed 20 days after inoculation. Although the cells of the vegetative apex still seem to be mitotically quiescent, procambial strands, as compared with those of the untreated seed in Fig. 2, are more evident (Fig. 5). In addition, waved cell walls occur, suggesting that the divisions of cells of the vegetative apex may begin soon (Fig. 6).
Fig. 5. Longitudinal section of a 2 0 - d a y - o l d embryo showing that procambial strands become more evident, as compared with the control in Fig. 2. p s = procambial strands; v = vegetative a p e x .
Fig. 6. Transverse section of a 2 0 - d a y - o l d embryo showing the waved cell walls of vegetative apex cells, v = v e g e t a t i v e a p e x .
166
Certain cells of the vegetative apex begin to divide 30 days after inoculation; their divisions are particularly observed in the cells which adjoin procambial strands and also in those of the deeper layers (Fig. 7). It is in the germinated seed that the real cell divisions of the vegetative apex start, and
Fig. 7. L o n g i t u d i n a l s e c t i o n o f a 3 0 - d a y - o l d e m b r y o s h o w i n g t h e first cell divisions o f t h e vegetative apex. e = e p i d e r m i s ; v = vegetative apex.
Fig. 8. L o n g i t u d i n a l s e c t i o n o f a 4 6 - d a y - o l d e m b r y o j u s t a f t e r g e r m i n a t i o n , s h o w i n g t h e real cell divisions o f t h e vegetative a p e x a n d t h e m o r e e v i d e n t p r o c a m b i a l strands, ps = p r o c a m b i a l s t r a n d s ; v = vegetative apex.
167 the first p r o t o x y l e m elements appear in their procambial strands (Figs. 8 and 9). During a period of 30--46 days, the anticlinal and periclinal cell divisions take place in the deeper layers of the apicies much more predominantly than the anticlinal divisions in the peripheral layers, b u t cell divisions are scarcely detectable in the embryos of seed cultured at 20°C for 40 days (Fig. 10).
Fig. 9. Transverse section of a 46-day-old embryo showing the cell divisions of the vegetative apex and the first protoxylem, px = protoxylem; v = vegetative apex.
After 60 days from inoculation, the anticlinal divisions in the peripheral layers of the vegetative apex, as well as the anticlinal or periclinal divisions in the deeper layers, occur more frequently and the mass of cells can distinctly be detected as a vegetative apex, b u t it is still flat (Fig. 11). The vegetative apex continuously increases in volume by both anticlinal and periclinal cell divisions and becomes a slightly d o m e d apex consisting of cells with dense cytoplasm and large nuclei, although procambial strands relating to the initiation of a leaf primordium are n o t observable in the apex at this stage (Figs. 12 and 13). On the other hand, such cell divisions and histological changes of the vegetative apex are scarcely observed in the embryos of seed cultured at 20°C for 70 days (Fig. 14).
168
Fig. 10. Longitudinal section of a 40-day-old embryo cultured at 20°C, showing the very limited extent of cell divisions of the vegetative apex and a small quantity of insoluble carbohydrates, ps = procambial strands; v = vegetative apex.
Fig. 11. Longitudinal section of a 60-day-old seedling showing the anticlinal or periclinal divisions in the deeper layers of a nearly fiat vegetative apex. e = epidermis; px = protoxylem; v = vegetative apex.
169
!i,f+~.~
Im
i0
f-
Fig. 12. Longitudinal s e c t i o n o f a 7 0 - d a y - o l d seedling s h o w i n g a slightly d o m e d a p e x consisting o f cells w i t h dense c y t o p l a s m and large nuclei.
Fig. 13. Transverse s e c t i o n o f a 7 0 - d a y - o l d seedling s h o w i n g an a l m o s t circular vegetative a p e x and vascular bundles, p x = p r o t o x y l e m ; v = vegetative a p e x ; vb = vascular bundles.
170
Fig. 14. Longitudinal section of a 70-day-old embryo cultured at 20°C, showing the very extent of cell divisions o f a vegetative apex. e = e p i d e r m i s ; v ffi v e g e t a t i v e a p e x .
limited
DISCUSSION L o w temperatures for the germination of tulip seeds in vivo are required for a b o u t 40--50 days (Sisa and Higuchi, 1967). The present results show that the tulip e m b r y o elongates within the seed at first and its radicle starts to emerge 40 days after inoculation. Thus, the low-temperature requirement for germination of the seeds of Tulipa gesneriana seems to be satisfied in 40--50 days. When germinated seeds with 1 to 2-mm long radicles are transferred to warm conditions at 20°C (Fig. 1C), all elongate their cotyledons upward, b u t only t w o fifths of these seedlings form bulbs (unpublished data). This result agrees fairly closely with that of a previous paper (Niimi, 1978). Thus, it seems that the d o r m a n c y of the tulip seed is ended as soon as the radicle appears o u t of the seed coat, b u t a longer duration of low temperature is necessary for the undifferentiated vegetative apex to develop fully. This point ~s also clarified by the present histological observations on the
171
development of the vegetative apex; the real cell divisions of the undifferentiated vegetative apex activated by low temperatures begin with the seed germination and the flat apex develops into a slightly domed one by 70 days from inoculation. These results also show that a relatively longer period of low temperatures is required for the full development of the vegetative apex. CONCLUSION
When the tulip seed is naturally dispersed from the capsule, the embryo is still immature because the vegetative apex is not fully developed. The cells of the undifferentiated apex are activated by low temperatures and the real divisions begin after seed germination. Thus, low temperatures affect the further differentiation of the vegetative apex as well as the induction of seed germination. ACKNOWLEDGEMENTS
The author is grateful to the Emeritus Professor Dr. M. Kumazawa of Nagoya University and to Professor Dr. T. Takano of Meijo University for valuable discussion and advice.
REFERENCES Feder, N. and O'Brien, T.P., 1968. Plant microtechnique: some principles and new methods. Am. J. Bot., 55: 123--142. Gaff, D.E., Zee, S.-Y. and O'Brien, T.P., 1976. The fine structure of dehydrated and reviving leaves of Borya nitida Lalill. -- a desiccation tolerant plant. Aust. J. Bot., 24: 225--236. Niimi, Y., 1978. Influence of low and high temperatures on the initiation and the development o f a bulb primordium in isolated tulip embryos. Scientia Hortic., 9: 61--69. Nitsch, J.P., 1951. Growth and development in vitro of excised ovaries. Am. J. Bot., 38: 566--577. Sidman, R.L., Mottla, P.A. and Feder, N., 1961. Improved polyester wax embedding for histology. Stain Technol., 36: 279--284. Sisa, M. and Higuchi, H., 1967. Studies on shortening the juvenile phase of tulips under controlled environments. I. On the germination of seed and growth of seedling. Jpn. J. Breed., 17: 122--130. (In Japanese with English summary.) Spurt, A.R., 1969. A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res., 26: 31--43.