Plant Cell Reports
Plant Cell Reports (1986) 5:119-123
© Springer-Verlag 1986
Cytochemical studies of callus development from microspore in cultured anther of rice S. S. Tsay 1, H. S. Tsay 2, and C.Y. Chao 1 1 Department of Biology, Tunghai University, Taichung, Taiwan 400, ROC 2 Department of Agronomy, Taiwan Agricultural Research Institute, Taichung, Taiwan 431, ROC Received August 12, 1985 / Revised version received January 13, 1986 - Communicated by J. M. Widholm
ABSTRACT Cytochemical studies of androgenic anthers of Oryza sativa picked from the culture at 2 day i n t e r vals from 0 to 40 days have been carried out. Glutaradehyde-OsO4-fixed and plastic-embedded sections were stained with TBO, SBB and PAS for acidic polymers, l i p i d s and polysaccharides respectively. Among the population only 4% of microspores, which accumulate abundant amorphous l i p i d in the f i r s t few days of culture, are androgenic. Less than 30%, which have many l i p i d granules and some amorphous l i p i d , become n u t r i t i v e microspores. Starch grains also accumulate in these n u t r i t i v e microspores which degenerate at the stage when the androgenic m u l t i c e l l u l a r microspores are in rapid development. The remaining microspores, which have no or l i t t l e l i p i d , degenerate early. At about the lO0-cell stage, each multicel l u l a r unit consists of two cell types, large and small. The large c e l l s contain abundant amorphous l i p i d and starch grains which the small ones stain intensely with TBO. Our results indicate that the epidermis and endothecium of the cultured anthers are not quiescent. They can accumulate and transport l i p i d and polysaccharides at certain stages during the cultural period. Globular embryoid appearing structures and l e a f - l i k e protrusions can be observed at the surface of the callus in about 40-day old culture, indicating that both embryogenesis and organogenesis may take place in rice callus. ABBREVIATIONS Os04; osmium tetroxide, TBO; toluidine blue O, SBB; sudan black B, PAS; periodic a c i d - S c h i f f ' s reagent, NAA; l-naphthaleneacetic acid. INTRODUCTION The induction of uninucleate microspores in cultured anthers to produce haploid plants has been successful in a number of plant species (see Bajaj 1983) The androgenic microspore can give r i s e either direct l y to a p l a n t l e t or to a callus from which plantlets can be induced to d i f f e r e n t i a t e . The mode of early d i v i s i o n of the androgenic microspore has been studied in Datura (Sunderland et a l . 1974), tobacco (Sunderland and Wicks 1971), ~ce-(Chen and Wu 1983) and other plants. Usually the f i r s t d i v i s i o n of the microspore produces a vegetative and a generative c e l l . However, v a r i a t i o n in the pathways to androgenesis can occur even in a single species (Sunderland et alo Offprint requests to: C.Y. Chao
1974; Chen and Wu 1983). In many plant species, the vegetative cell takes part in further development. In a few species, such as Hyoscyamus ~ (henbane), the generative cell develops further (Raghavan 1976). During anther culture, only a small number of the uninucleate microspores are androgenic and the majority of them are not. Several investigators have attempted to d i f f e r e n t i a t e between the androgenic and nonandrogenic microspores during i n i t i a t i o n by cytochemical means. In henbane, Raghavan (1979a, b, 1981) reported that androgenic microspores can synthesize RNA as early as I-2 h a f t e r inoculation while the nonandrogenic ones can not. Sangawan and Norreel (1979) previously reported s i m i l a r findings in Datura. In the experiments reported here, we examine the deposition of two nutrients, namely l i p i d s and polysaccharides, necessary for callus development from the microspore in cultured rice anthers. Evidence is also presented for the involvements of l i p i d s in the induction of the androgenic microspores. MATERIAL AND METHODS Oryza sativa var. Tainung 67 plants were f i e l d grown at the Taiwan Agricultural Research I n s t i t u t e . Panicles at the appropriate stage were collected from donor material and anthers containing uninucleate microspores were cultured according to the procedures described by Chen (1977). A medium composed of N~ inorganic salts (Chu et a l . 1975), and MS organic~sub stances (Murashige and Skoog 1962) supplemented with 4 mg/l NAA and 2 mg/l kinetin (Chen et a l . 1982). The cultures were incubated in the d ~ k ~ t about 26°C. In general, 20 anthers were removed from culture tubes at 2 day i n t e r v a l s over a 40 day period and fixed in 6% cold glutaraldehyde in phosphate buffer (pH 7.2) for 4 h. They were then post fixed in 1% cold OsO~ overnight (Chao 1977). After dehydration, the anthers were embedded in Epon 812. Five anthers were then selected at each i n t e r v a l and sectioned on an u l t r a microtome at a thickness of 1 ~m. Sections were stained with 0.1% TBO in acetic buffer at pH 5.6(Chayen et a l . 1973). Acidic polymers are stained purplish-red (metachromatic) with this dye. Lipids were stained with a saturated solution of SBB in 70% ethanol (Chao 1979). Polysaccharides were stained with PAS (Pearse 1968). RESULTS 1. Callus development At the time of culture i n i t i a t i o n , the tapetal c e l l s in the anther stain moderately with TBO, i n d i -
120 Figs. 1-9. Portions of sections of cultured anthers stained with TBO. 1. Uninucleate microspores (double arrows) and tapetal c e l l s (single arrows) at the time of i n o c u l a t i o n . Note many dark granu~ les in tapetal c e l l s (300 X). 2. A microspore containing a vegetative nucleus (double arrows) and a generative uncleus (single arrow) in a 4-day old culture (750 X). 3. A multinucleate microspore (arrow) in a 6-day old c u l t u r e (300 X). 4. Cell walls being formed in a multinucleate microspore (arrow) I0 days a f t e r culture initiation. Note several degenerated microspores (300 X). 5. A microspore (arrow) containing 2 nuclei derived from the generative nucleus in a 10day old c u l t u r e (300 X). 6. Two m u l t i c e l l u l a r microspores (arrows) together with several degenerated ones in an 18-day old c u l t u r e (150 X). 7. A m u l t i c e l l u l a r microspore containing several large c e l l s (L) and many small ones (S) 28 days a f t e r i n o c u l a t i o n . Note d i f f e r e n t s t a i n a b i l i t y of these two types of c e l l s (300 X). 8o A globular embryoid (arrow) at the surface of callus in a 32-day old c u l t u r e . Double arrows indicate a group of colorless c o l l e n chyma c e l l s at the center (75 X). 9. A callus consisting of a peripheral (P) and a central (C) region in a 36-days old c u l t u r e . Note the protrusions (arrows) at the callus surface (75 X).
7
6
cating that they contain some acidic substances (Fig. I ) . Many i n c l u s i o n bodies are present in t h e i r cytoplasm (Fig. I ) . These bodies were shown to be l i p i d a f t e r SBB s t a i n i n g . The pattern of early development of the androgenic microspores observed in our TBO preparations was s i m i l a r to that reported by Chen and Wu (1983). The f i r s t pollen mitosis takes place w i t h i n 4 days a f t e r i n o c u l a t i o n , r e s u l t i n g in the formation of a vegetat i v e and a generative nucleus (Fig. 2), or, in some cases, two s i m i l a r nuclei. The wall between the two daughter nuclei may or may not be formed. In many androgenic microspores, the vegetative nucleus or c e l l can divide f u r t h e r to form a multinucleate (Fig. 3) or m u l t i c e l l u l a r (Fig. 4) u n i t . In a few cases, however, the generative nucleus (Fig. 5) or 2 s i m i l a r nuclei nay d i v i d e . In 2-3 week old cultures, the androgenic microspores are m u l t i c e l l u l a r and each consists of about 30 c e l l s (Fig. 6). As the cell number increases to about lOO during the next week, two types of c e l l s appear to d i f f e r e n t i a t e , with each u n i t consisting of several large and l i g h t l y stained c e l l s and many small and darkly stained ones (Fig. 7). Young c a l l i liberated from the spore exines are
present in anther locules in 5 week old cultures. Each callus seems to consist of s i m i l a r c e l l types at f i r s t , however c e l l d i f f e r e n t i a t i o n soon follows. A globular embryoid attached to a callus is shown in Figure 8 (single arrow). Double arrows in this f i gure point to a group of large and l i g h t l y stained c e l l s with unevenly thickened primary walls at the center of the c a l l u s . They were i d e n t i f i e d as collenchyma c e l l s . Figure 9 shows a callus consisting of a peripheral and central region. Cells in these two regions are somewhat d i f f e r e n t in size and s t a i n a b i l i t y with TBO. The same f i g u r e also shows that several l e a f - l i k e protrusions appear to form at the callus surface. 2. Changes in concentration and d i s t r i b u t i o n of l i p i d s f u r i n g androgenesis At the time of anther culture i n i t i a t i o n , very l i t t l e stainable l i p i d is present in the epidermis, endothecium and microspores. The tapetal c e l l s , which are i r r e g u l a r in shape and in close contact with the microspores, contain abundant granular and some amorphous l i p i d s (Fig. I 0 ) . A f t e r 4 days of c u l t u r e , however, the state of l i p i d substances in the anther is altered considerably. Both the epi-
121 Figs. 10-16. Portions of sections of cultured anthers stained with SBB. i0. Uninucleate microspores (double arrows) and tapetal c e l l s (single arrows) at the time of i n o c u l a t i o n . Note the l i p i d granules in the l a t t e r (300 ×). 11. Four types of microspores ( I - I V ) can be c l a s s i f i e d according to the l i p i d content in the 4-day old c u l t u r e (300 X). 12. An androgenic microspore (single arrow) and a n u t r i t i v e microspore (double arrows) along with several degenerated ones in an 18-day old c u l t u r e (300 X). 13. A m u l t i c e l l u l a r microspore (MM) cont a i n i n g starch grains (as b r i g h t spots) and surrounded by d i f f u s e l i p i d (single arrows) in a 22-day old c u l t u r e . Note the starch grains in anther wall (double arrows) (300 X). 14. A m u l t i c e l l u l a r microspore consist i n g of several large (L) and many small (S) c e l l s in a 28-day old c u l t u r e . Note d i f f e r e n t c o l o r a t i o n of two types of c e l l s (300 X). 15. A callus containing abundant amorphous l i p i d and some small starch grains 32 days a f t e r i n o c u l a t i o n (150 X). 16. A callus in a 36-day old c u l t u r e . Note the protrusion (single arrow) and globular embryoid (double arrows) (150
×).
dermis and endothecium contain some amorphous l i p i d now. Tapetal c e l l s are degenerating. Their contents intrude among the microspores (Fig. I I ) to form the so-called tryphine, whose main component is l i p i d (Esau1977). The microspores themselves have now accumulated various amounts of l i p i d . Depending on the degree of s t a i n a b i l i t y with SBB 4 types of microspores (Fig. I I ) can be i d e n t i f i e d at t h i s stage: Type I . Microspores are l i g h t gray in color, i n d i cating the absence of l i p i d . From the t o t a l populat i o n , c a . 51% of the microspores belong to t h i s type. Type I I . Microspores are darkly stained due to the presence of a great amount of amorphous l i p i d . Upon close~amination, i t is revealed that each of these microspores contains two c e l l s or nuclei. Less than 4% of the microspores are of t h i s type. Type I I I . Microspores contain both amorphous and granular l i p i d s . About 30% microspores belong to t h i s type. Type IV. Microspores contain only a few l i p i d granules. The remaining 15% of the microspores belong to t h i s type. From the observations of a l l our preparations, we can draw the f o l l o w i n g conclusions: Type I and IV microspores are nonandrogenic and eventually degenerate, Type I I microspores are androgenic. They can develop into m u l t i c e l l u l a r units and eventually form calli. Microspores of Type I I I are thought to provide n u t r i t i v e f u n c t i o n . They can store l i p i d s and polysaccharides f o r up to 2-3 weeks a f t e r which they also degenerate, Their stored n u t r i e n t s may be one of the main sources of nourishment for the developing
m u l t i c e l l u l a r masses. Figure 12 shows an androgenic (single arrow) and n u t r i t i v e (double arrows) microspore together with several degenerating ones in an 18day old c u l t u r e . Several days l a t e r the androgenic microspores have acquired many large starch grains which appear as b r i g h t spots in the SBB preparations (Fig. 13). Starch grains also appear in the epidermal and endothecial c e l l s at t h i s stage (Fig. 13)o At the same time the n u t r i t i v e microspores begin to degenerate and t h e i r remains can be found around the androgenic ones, each of which is now composed of about 30 c e l l s (Fig. 13). As in the TBO preparations, two types of c e l l s can be observed in each of the m u l t i c e l l u l a r microspores in about 4-week old cultures (Fig. 14). However, the large c e l l s are colored much more intensely with SBB than the small c e l l s , i n d i c a t i n g that the former contain more l i p i d than the l a t e r . Most of the starch grains in the androgenic microspores as well as in the anther wall have disappeared at t h i s stage. The young callus released from the spore exine contains a large amount of amorphous l i p i d evenly d i s t r i b u t e d among the c e l l s which are more or less uniform in size at f i r s t (Fig. 15). L e a f - l i k e protrusions and globular embryoids soon develop at the surface of the callus (Fig. 16). In both TBO (Fig. 9) and SBB (Fig. 16) preparations, the protrusions cons i s t of r e l a t i v e l y large and l i g h t l y stained c e l l s . 3. Change in concentration and d i s t r i b u t i o n of polysaccharides during androgenesis A very low concentration of polysaccharides can
122 Figs. 17-24. Portions of sections of cultured anthers stained with PAS. 17. An anther at the time of inoculation indicating the epidermis (EP), endothecium (E), tapetum (T) and uninucleate microspores (UM). Note that only tapetal cells contain some amorphous polysaccharides (750 X). 18, A multinucleate microspore (single arrow) containing the generative nucleus and several small nuclei derived from the vegetative nucleus in a 6-day old culture. Double arrows indicate the wall between the vegetative and generative cell in a microspore. Note that most tapetal cells have been l y sed (750 X). 19, A m u l t i c e l l u l a r microspore (single arrow) and a n u t r i t i v e microspore ( double arrows), both containing starch grains, I0 days a f t e r culture i n i t i a tion (750 X). 20. A m u l t i c e l l u l a r microspore consisting of several large (L) and many smal l (S) c e l l s 22 days a f t e r the inoculation. Large c e l l s contain starch grains while small ones have some amorphous polysaccharides. Note some d i ffuse polysaccharides (arrows) around the m u l t i c e l l u l a r microspore liberated from the degenerated microspores (300
X),
21, A young callus containing many small starch grains in a 36-day old culture (150 X). 22. An older callus containing many large and small starch grains 38 days a f t e r the inoculation (75 X). 23, Portion of a callus of Fig. 22 showing the protrusions (arrows) are being formed at the surface (150 X). 24. A globular embryoid (arrow) at the surface of a callus in a 38-day old culture (300 X).
be seen by staining with PAS in the various types of c e l l s in the anther at the time of inoculation except for some diffuse material in the tapetal c e l l s (Fig. 17). Following the formation of the cell wall between the vegetative and generative uncleus (Fig. 18), small starch grains begin to accumulate in the androgenic microspores. Large grains then follow. Figure 19 shows a m u l t i c e l l u l a r microspore (single arrow} and a n u t r i t i v e one (double arrows), both containing small and large starch grains in a lO-day old culture. Figure 20 shows an androgenic microspore consisting of several large and many small cells in a 22-day culture. As shown in the f i g u r e , only the two large c e l l s in this section have starch grains. The same figure contains some degenerating n u t r i t i v e microspores l i b e r a t i n g t h e i r contents around the androgenic one. Young callus contains small starch grains (Fig. 21). While large grains soon accumulate and d i f f e r entiating callus is f i l l e d with many starch grains (Fig. 22). The l e a f - l i k e protrusions and embryoidl i k e structures (Figs. 23 and 24) at the surface of the callus can be seen more c l e a r l y in the PAS preparations than in the TBO and SBB preparations.
DISCUSSION At the time of inoculation, the anther wall of rice consists of the epidermis, endothecium and tapetum. Very l i t t l e l i p i d and polysaccharide are present in the former two tissues. The tapetal c e l l s , which begin to degenerate at this stage, contain many l i p i d granules and some amorphous polysaccharide. Very l i t t l e l i p i d and polysaccharide can be found in the vacuolated uninucleate microspores. In the f i r s t few days of culture, the microspores obtain t h e i r nutrients mainly from the degenerating tapetal c e l l s . The amount and kinds of l i p i d s which the microspores obtain can be variable. Based on the l i p i d content, we c l a s s i f y and characterize four types of microspores in the 4-day old cultures. Among the population of microspores, only about 4% (Type I I ) with abundant amorphous l i p i d are androgenic and capable of dividing and forming m u l t i c e l l u l a r units. Less than 30% of the microspores (Type I I I ) are transformed into n u t r i t i v e ones f o r storing l i p i d granules and starch grains. Their nucleus may divide but can not form m u l t i c e l l u l a r units. The remaining microspores (Type I and IV) with very l i t t l e l i p i d degener-
123 ate sooner or l a t e r . Thus the a b i l i t y of the uninucleate microspores to synthesize and u t i l i z e l i p i d s at t h e i r early developmental stage seems to be one of the decisive factors in switching from the gametophytic to sporophytic development. Raghavan (1979 a, b, 1981) reported that in henbane embryogenic microspores can synthesize RNA in the f i r s t hour of culture. Presumably carbohydrates may have a simil a r role in this respect However, this could not be wholly studied in-our'PAS preparations due to the fact that most of the soluble carbohydrates were washed o f f at the time of f i x a t i o n and dehydration. This warrents f u r t h e r study by using freeze-substituted, plastic-embedded anthers which can preserve much of the soluble carbohydrates (Chao 1977). Although the n u t r i t i v e microspores are not embryogenic. They can store l i p i d s , polysaccharides and possibly other substances. These c e l l s degenerate and apparently l i b e r a t e t h e i r stored food at the stage when the embryogenic microspores are in rapid development. Their contents then may be the main source of nutrients for the developing m u l t i c e l l u l a r units at this stage. Our results indicate that the epidermal and endothecial c e l l s of the cultured anthers of rice are not quiescent throughout the cultural period. At the time of culture i n i t i a t i o n , both tissue cells are devoid of l i p i d and pol~accharide. Amouphous l i p i d s appear in these tissues before long and starch grains can be observed in about 3-week old cultures, indicating that they are metabolically active. Thus, they can transport and store nutrients, much of which can reach the developing microsporeso The young c a l l i liberated from the spore wall contain a high concentration of amorphous l i p i d s and a moderate level of starch grains. At a l a t e r stage, they accumulate more of these substances. Cell d i f f e r e n t i a t i o n then takes place within each callus. Some peripheral c e l l s became the primordia of l e a f l i k e protrusions and embryoids. Since our observations were made from the materials collected within 40 days a f t e r the culture i n i t i a t i o n , no well organized embryoids were observed in our c a l l i . Genovesi and Magill (1982) reported that both organogenesis and embryogenesis can take place in rice c a l l i d e r i ved from microspores. The presence of l e a f - l i k e protrusions and globular structures at the surface of our c a l l i are in agreement w~th t h e i r findings. One i n t e r e s t i n g finding in our experiments should be mentioned here. At about the lO0-cell stage, each m u l t i c e l l u l a r microspore consists of two types of c e l l s , a large and small class. This d i f f e r e n t i a t i o n of c e l l s was observed with a l l three staining procedures. The large c e l l s are rich in amorphous l i p i d s and starch grains but stain l i g h t l y withTBO while the small ones have much less l i p i d and no starch grains but stained with TBO much more intensely. Thus, they seem to perform quite d i f f e r e n t functions, the former probably n u t r i t i v e and the l a t e r reproductive. A more detailed study of the d i f f e r e n t i a t i o n of these
two types of c e l l s , especially t h e i r ontogenetic relationship with the vegetative and generative c e l l , and t h e i r roles in the developing callus should be carried out before a d e f i n i t e conclusion can be reached. ACKNOWLEDGEMENTS We thank the National Science Council f o r f i nancial support. REFERENCES Bajaj YPS (1983) In:Evans, Sharp, Ammirato and Yamada (eds.), Handbook'-of Plant Cell Culture. Macmillan Pub. Co., New York. pp. 228-287. Bourgin JP, Nitsch JP (1967) Ann. Physiol. Veg. 9: 377-382. Chao CY (1977) Amer. J. Bot. 64:921-930. Chao CY (1979) Phytomorphology 29:381-387. Chayer J, Bitensky L, Butcher RG (1973) Practical Histochemistry. Wiley, London. Chen CC (1977) In Vitro 13:484-489. Chen CC, Wu YH (1983) Proc. Natl. Sci. Counc. Bo, ROC 7:151-157o Chen LJ, Lai PC, Liao CH, Tsay HS (1982) J. Agric. Res. China 31:283-290. Chu CC, Wang CC, Sun CS, Chen H, Yin KC, Chu CY, Bi FY (1975) Sin Sinica 18:659-668. Esau K (1977) John Wiley and Sons, New York. Genovesi AD, Magill CW (1982) Plant Cell Report I : 257-260. Guha S, Maheshwari SC (1964) Nature 204:497. Guha-Makherjie S (1973) J. Exp. Bot. 24:139-144. l y e r RD, Raina SK (1972) Planta 104:146-156. Murashige T, Skoog F (1962) Physiol. Plant. 15:473497. Niizeki H, Oono K (1968) Proc. Jap. Acad. 44:554-557. Pearse AGE (1968) Histochemistry, Theoretical and Applied. Vol. I . J. and A. Churchill, Ltd., London. Raghavan V (1976) Science 191:188-189. Raghavan V (1979a) Amer.J. Bot. 66:36-39. Raghaven V (1979b) Amer. J. Bot. 66:784-795. Raghaven V (1981) J. Cell Biol. 89:593-606. Sangwan-Norrell BS (1978) Can. J. Bot. 56:805-817. Sunderland N, Collins GB, Dunwell JM (1974) Planta 117:227-241. Sunderland N, Wicks FN (1971) J. Exp. Bot. 22:213-216.
Plant Cell Reports (1986) 5:119-123
© Springer-Verlag 1986
Cytochemical studies of callus development from microspore in cultured anther of rice S. S. Tsay 1, H. S. Tsay 2, and C.Y. Chao 1 1 Department of Biology, Tunghai University, Taichung, Taiwan 400, ROC 2 Department of Agronomy, Taiwan Agricultural Research Institute, Taichung, Taiwan 431, ROC Received August 12, 1985 / Revised version received January 13, 1986 - Communicated by J. M. Widholm
ABSTRACT Cytochemical studies of androgenic anthers of Oryza sativa picked from the culture at 2 day i n t e r vals from 0 to 40 days have been carried out. Glutaradehyde-OsO4-fixed and plastic-embedded sections were stained with TBO, SBB and PAS for acidic polymers, l i p i d s and polysaccharides respectively. Among the population only 4% of microspores, which accumulate abundant amorphous l i p i d in the f i r s t few days of culture, are androgenic. Less than 30%, which have many l i p i d granules and some amorphous l i p i d , become n u t r i t i v e microspores. Starch grains also accumulate in these n u t r i t i v e microspores which degenerate at the stage when the androgenic m u l t i c e l l u l a r microspores are in rapid development. The remaining microspores, which have no or l i t t l e l i p i d , degenerate early. At about the lO0-cell stage, each multicel l u l a r unit consists of two cell types, large and small. The large c e l l s contain abundant amorphous l i p i d and starch grains which the small ones stain intensely with TBO. Our results indicate that the epidermis and endothecium of the cultured anthers are not quiescent. They can accumulate and transport l i p i d and polysaccharides at certain stages during the cultural period. Globular embryoid appearing structures and l e a f - l i k e protrusions can be observed at the surface of the callus in about 40-day old culture, indicating that both embryogenesis and organogenesis may take place in rice callus. ABBREVIATIONS Os04; osmium tetroxide, TBO; toluidine blue O, SBB; sudan black B, PAS; periodic a c i d - S c h i f f ' s reagent, NAA; l-naphthaleneacetic acid. INTRODUCTION The induction of uninucleate microspores in cultured anthers to produce haploid plants has been successful in a number of plant species (see Bajaj 1983) The androgenic microspore can give r i s e either direct l y to a p l a n t l e t or to a callus from which plantlets can be induced to d i f f e r e n t i a t e . The mode of early d i v i s i o n of the androgenic microspore has been studied in Datura (Sunderland et a l . 1974), tobacco (Sunderland and Wicks 1971), ~ce-(Chen and Wu 1983) and other plants. Usually the f i r s t d i v i s i o n of the microspore produces a vegetative and a generative c e l l . However, v a r i a t i o n in the pathways to androgenesis can occur even in a single species (Sunderland et alo Offprint requests to: C.Y. Chao
1974; Chen and Wu 1983). In many plant species, the vegetative cell takes part in further development. In a few species, such as Hyoscyamus ~ (henbane), the generative cell develops further (Raghavan 1976). During anther culture, only a small number of the uninucleate microspores are androgenic and the majority of them are not. Several investigators have attempted to d i f f e r e n t i a t e between the androgenic and nonandrogenic microspores during i n i t i a t i o n by cytochemical means. In henbane, Raghavan (1979a, b, 1981) reported that androgenic microspores can synthesize RNA as early as I-2 h a f t e r inoculation while the nonandrogenic ones can not. Sangawan and Norreel (1979) previously reported s i m i l a r findings in Datura. In the experiments reported here, we examine the deposition of two nutrients, namely l i p i d s and polysaccharides, necessary for callus development from the microspore in cultured rice anthers. Evidence is also presented for the involvements of l i p i d s in the induction of the androgenic microspores. MATERIAL AND METHODS Oryza sativa var. Tainung 67 plants were f i e l d grown at the Taiwan Agricultural Research I n s t i t u t e . Panicles at the appropriate stage were collected from donor material and anthers containing uninucleate microspores were cultured according to the procedures described by Chen (1977). A medium composed of N~ inorganic salts (Chu et a l . 1975), and MS organic~sub stances (Murashige and Skoog 1962) supplemented with 4 mg/l NAA and 2 mg/l kinetin (Chen et a l . 1982). The cultures were incubated in the d ~ k ~ t about 26°C. In general, 20 anthers were removed from culture tubes at 2 day i n t e r v a l s over a 40 day period and fixed in 6% cold glutaraldehyde in phosphate buffer (pH 7.2) for 4 h. They were then post fixed in 1% cold OsO~ overnight (Chao 1977). After dehydration, the anthers were embedded in Epon 812. Five anthers were then selected at each i n t e r v a l and sectioned on an u l t r a microtome at a thickness of 1 ~m. Sections were stained with 0.1% TBO in acetic buffer at pH 5.6(Chayen et a l . 1973). Acidic polymers are stained purplish-red (metachromatic) with this dye. Lipids were stained with a saturated solution of SBB in 70% ethanol (Chao 1979). Polysaccharides were stained with PAS (Pearse 1968). RESULTS 1. Callus development At the time of culture i n i t i a t i o n , the tapetal c e l l s in the anther stain moderately with TBO, i n d i -
120 Figs. 1-9. Portions of sections of cultured anthers stained with TBO. 1. Uninucleate microspores (double arrows) and tapetal c e l l s (single arrows) at the time of i n o c u l a t i o n . Note many dark granu~ les in tapetal c e l l s (300 X). 2. A microspore containing a vegetative nucleus (double arrows) and a generative uncleus (single arrow) in a 4-day old culture (750 X). 3. A multinucleate microspore (arrow) in a 6-day old c u l t u r e (300 X). 4. Cell walls being formed in a multinucleate microspore (arrow) I0 days a f t e r culture initiation. Note several degenerated microspores (300 X). 5. A microspore (arrow) containing 2 nuclei derived from the generative nucleus in a 10day old c u l t u r e (300 X). 6. Two m u l t i c e l l u l a r microspores (arrows) together with several degenerated ones in an 18-day old c u l t u r e (150 X). 7. A m u l t i c e l l u l a r microspore containing several large c e l l s (L) and many small ones (S) 28 days a f t e r i n o c u l a t i o n . Note d i f f e r e n t s t a i n a b i l i t y of these two types of c e l l s (300 X). 8o A globular embryoid (arrow) at the surface of callus in a 32-day old c u l t u r e . Double arrows indicate a group of colorless c o l l e n chyma c e l l s at the center (75 X). 9. A callus consisting of a peripheral (P) and a central (C) region in a 36-days old c u l t u r e . Note the protrusions (arrows) at the callus surface (75 X).
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cating that they contain some acidic substances (Fig. I ) . Many i n c l u s i o n bodies are present in t h e i r cytoplasm (Fig. I ) . These bodies were shown to be l i p i d a f t e r SBB s t a i n i n g . The pattern of early development of the androgenic microspores observed in our TBO preparations was s i m i l a r to that reported by Chen and Wu (1983). The f i r s t pollen mitosis takes place w i t h i n 4 days a f t e r i n o c u l a t i o n , r e s u l t i n g in the formation of a vegetat i v e and a generative nucleus (Fig. 2), or, in some cases, two s i m i l a r nuclei. The wall between the two daughter nuclei may or may not be formed. In many androgenic microspores, the vegetative nucleus or c e l l can divide f u r t h e r to form a multinucleate (Fig. 3) or m u l t i c e l l u l a r (Fig. 4) u n i t . In a few cases, however, the generative nucleus (Fig. 5) or 2 s i m i l a r nuclei nay d i v i d e . In 2-3 week old cultures, the androgenic microspores are m u l t i c e l l u l a r and each consists of about 30 c e l l s (Fig. 6). As the cell number increases to about lOO during the next week, two types of c e l l s appear to d i f f e r e n t i a t e , with each u n i t consisting of several large and l i g h t l y stained c e l l s and many small and darkly stained ones (Fig. 7). Young c a l l i liberated from the spore exines are
present in anther locules in 5 week old cultures. Each callus seems to consist of s i m i l a r c e l l types at f i r s t , however c e l l d i f f e r e n t i a t i o n soon follows. A globular embryoid attached to a callus is shown in Figure 8 (single arrow). Double arrows in this f i gure point to a group of large and l i g h t l y stained c e l l s with unevenly thickened primary walls at the center of the c a l l u s . They were i d e n t i f i e d as collenchyma c e l l s . Figure 9 shows a callus consisting of a peripheral and central region. Cells in these two regions are somewhat d i f f e r e n t in size and s t a i n a b i l i t y with TBO. The same f i g u r e also shows that several l e a f - l i k e protrusions appear to form at the callus surface. 2. Changes in concentration and d i s t r i b u t i o n of l i p i d s f u r i n g androgenesis At the time of anther culture i n i t i a t i o n , very l i t t l e stainable l i p i d is present in the epidermis, endothecium and microspores. The tapetal c e l l s , which are i r r e g u l a r in shape and in close contact with the microspores, contain abundant granular and some amorphous l i p i d s (Fig. I 0 ) . A f t e r 4 days of c u l t u r e , however, the state of l i p i d substances in the anther is altered considerably. Both the epi-
121 Figs. 10-16. Portions of sections of cultured anthers stained with SBB. i0. Uninucleate microspores (double arrows) and tapetal c e l l s (single arrows) at the time of i n o c u l a t i o n . Note the l i p i d granules in the l a t t e r (300 ×). 11. Four types of microspores ( I - I V ) can be c l a s s i f i e d according to the l i p i d content in the 4-day old c u l t u r e (300 X). 12. An androgenic microspore (single arrow) and a n u t r i t i v e microspore (double arrows) along with several degenerated ones in an 18-day old c u l t u r e (300 X). 13. A m u l t i c e l l u l a r microspore (MM) cont a i n i n g starch grains (as b r i g h t spots) and surrounded by d i f f u s e l i p i d (single arrows) in a 22-day old c u l t u r e . Note the starch grains in anther wall (double arrows) (300 X). 14. A m u l t i c e l l u l a r microspore consist i n g of several large (L) and many small (S) c e l l s in a 28-day old c u l t u r e . Note d i f f e r e n t c o l o r a t i o n of two types of c e l l s (300 X). 15. A callus containing abundant amorphous l i p i d and some small starch grains 32 days a f t e r i n o c u l a t i o n (150 X). 16. A callus in a 36-day old c u l t u r e . Note the protrusion (single arrow) and globular embryoid (double arrows) (150
×).
dermis and endothecium contain some amorphous l i p i d now. Tapetal c e l l s are degenerating. Their contents intrude among the microspores (Fig. I I ) to form the so-called tryphine, whose main component is l i p i d (Esau1977). The microspores themselves have now accumulated various amounts of l i p i d . Depending on the degree of s t a i n a b i l i t y with SBB 4 types of microspores (Fig. I I ) can be i d e n t i f i e d at t h i s stage: Type I . Microspores are l i g h t gray in color, i n d i cating the absence of l i p i d . From the t o t a l populat i o n , c a . 51% of the microspores belong to t h i s type. Type I I . Microspores are darkly stained due to the presence of a great amount of amorphous l i p i d . Upon close~amination, i t is revealed that each of these microspores contains two c e l l s or nuclei. Less than 4% of the microspores are of t h i s type. Type I I I . Microspores contain both amorphous and granular l i p i d s . About 30% microspores belong to t h i s type. Type IV. Microspores contain only a few l i p i d granules. The remaining 15% of the microspores belong to t h i s type. From the observations of a l l our preparations, we can draw the f o l l o w i n g conclusions: Type I and IV microspores are nonandrogenic and eventually degenerate, Type I I microspores are androgenic. They can develop into m u l t i c e l l u l a r units and eventually form calli. Microspores of Type I I I are thought to provide n u t r i t i v e f u n c t i o n . They can store l i p i d s and polysaccharides f o r up to 2-3 weeks a f t e r which they also degenerate, Their stored n u t r i e n t s may be one of the main sources of nourishment for the developing
m u l t i c e l l u l a r masses. Figure 12 shows an androgenic (single arrow) and n u t r i t i v e (double arrows) microspore together with several degenerating ones in an 18day old c u l t u r e . Several days l a t e r the androgenic microspores have acquired many large starch grains which appear as b r i g h t spots in the SBB preparations (Fig. 13). Starch grains also appear in the epidermal and endothecial c e l l s at t h i s stage (Fig. 13)o At the same time the n u t r i t i v e microspores begin to degenerate and t h e i r remains can be found around the androgenic ones, each of which is now composed of about 30 c e l l s (Fig. 13). As in the TBO preparations, two types of c e l l s can be observed in each of the m u l t i c e l l u l a r microspores in about 4-week old cultures (Fig. 14). However, the large c e l l s are colored much more intensely with SBB than the small c e l l s , i n d i c a t i n g that the former contain more l i p i d than the l a t e r . Most of the starch grains in the androgenic microspores as well as in the anther wall have disappeared at t h i s stage. The young callus released from the spore exine contains a large amount of amorphous l i p i d evenly d i s t r i b u t e d among the c e l l s which are more or less uniform in size at f i r s t (Fig. 15). L e a f - l i k e protrusions and globular embryoids soon develop at the surface of the callus (Fig. 16). In both TBO (Fig. 9) and SBB (Fig. 16) preparations, the protrusions cons i s t of r e l a t i v e l y large and l i g h t l y stained c e l l s . 3. Change in concentration and d i s t r i b u t i o n of polysaccharides during androgenesis A very low concentration of polysaccharides can
122 Figs. 17-24. Portions of sections of cultured anthers stained with PAS. 17. An anther at the time of inoculation indicating the epidermis (EP), endothecium (E), tapetum (T) and uninucleate microspores (UM). Note that only tapetal cells contain some amorphous polysaccharides (750 X). 18, A multinucleate microspore (single arrow) containing the generative nucleus and several small nuclei derived from the vegetative nucleus in a 6-day old culture. Double arrows indicate the wall between the vegetative and generative cell in a microspore. Note that most tapetal cells have been l y sed (750 X). 19, A m u l t i c e l l u l a r microspore (single arrow) and a n u t r i t i v e microspore ( double arrows), both containing starch grains, I0 days a f t e r culture i n i t i a tion (750 X). 20. A m u l t i c e l l u l a r microspore consisting of several large (L) and many smal l (S) c e l l s 22 days a f t e r the inoculation. Large c e l l s contain starch grains while small ones have some amorphous polysaccharides. Note some d i ffuse polysaccharides (arrows) around the m u l t i c e l l u l a r microspore liberated from the degenerated microspores (300
X),
21, A young callus containing many small starch grains in a 36-day old culture (150 X). 22. An older callus containing many large and small starch grains 38 days a f t e r the inoculation (75 X). 23, Portion of a callus of Fig. 22 showing the protrusions (arrows) are being formed at the surface (150 X). 24. A globular embryoid (arrow) at the surface of a callus in a 38-day old culture (300 X).
be seen by staining with PAS in the various types of c e l l s in the anther at the time of inoculation except for some diffuse material in the tapetal c e l l s (Fig. 17). Following the formation of the cell wall between the vegetative and generative uncleus (Fig. 18), small starch grains begin to accumulate in the androgenic microspores. Large grains then follow. Figure 19 shows a m u l t i c e l l u l a r microspore (single arrow} and a n u t r i t i v e one (double arrows), both containing small and large starch grains in a lO-day old culture. Figure 20 shows an androgenic microspore consisting of several large and many small cells in a 22-day culture. As shown in the f i g u r e , only the two large c e l l s in this section have starch grains. The same figure contains some degenerating n u t r i t i v e microspores l i b e r a t i n g t h e i r contents around the androgenic one. Young callus contains small starch grains (Fig. 21). While large grains soon accumulate and d i f f e r entiating callus is f i l l e d with many starch grains (Fig. 22). The l e a f - l i k e protrusions and embryoidl i k e structures (Figs. 23 and 24) at the surface of the callus can be seen more c l e a r l y in the PAS preparations than in the TBO and SBB preparations.
DISCUSSION At the time of inoculation, the anther wall of rice consists of the epidermis, endothecium and tapetum. Very l i t t l e l i p i d and polysaccharide are present in the former two tissues. The tapetal c e l l s , which begin to degenerate at this stage, contain many l i p i d granules and some amorphous polysaccharide. Very l i t t l e l i p i d and polysaccharide can be found in the vacuolated uninucleate microspores. In the f i r s t few days of culture, the microspores obtain t h e i r nutrients mainly from the degenerating tapetal c e l l s . The amount and kinds of l i p i d s which the microspores obtain can be variable. Based on the l i p i d content, we c l a s s i f y and characterize four types of microspores in the 4-day old cultures. Among the population of microspores, only about 4% (Type I I ) with abundant amorphous l i p i d are androgenic and capable of dividing and forming m u l t i c e l l u l a r units. Less than 30% of the microspores (Type I I I ) are transformed into n u t r i t i v e ones f o r storing l i p i d granules and starch grains. Their nucleus may divide but can not form m u l t i c e l l u l a r units. The remaining microspores (Type I and IV) with very l i t t l e l i p i d degener-
123 ate sooner or l a t e r . Thus the a b i l i t y of the uninucleate microspores to synthesize and u t i l i z e l i p i d s at t h e i r early developmental stage seems to be one of the decisive factors in switching from the gametophytic to sporophytic development. Raghavan (1979 a, b, 1981) reported that in henbane embryogenic microspores can synthesize RNA in the f i r s t hour of culture. Presumably carbohydrates may have a simil a r role in this respect However, this could not be wholly studied in-our'PAS preparations due to the fact that most of the soluble carbohydrates were washed o f f at the time of f i x a t i o n and dehydration. This warrents f u r t h e r study by using freeze-substituted, plastic-embedded anthers which can preserve much of the soluble carbohydrates (Chao 1977). Although the n u t r i t i v e microspores are not embryogenic. They can store l i p i d s , polysaccharides and possibly other substances. These c e l l s degenerate and apparently l i b e r a t e t h e i r stored food at the stage when the embryogenic microspores are in rapid development. Their contents then may be the main source of nutrients for the developing m u l t i c e l l u l a r units at this stage. Our results indicate that the epidermal and endothecial c e l l s of the cultured anthers of rice are not quiescent throughout the cultural period. At the time of culture i n i t i a t i o n , both tissue cells are devoid of l i p i d and pol~accharide. Amouphous l i p i d s appear in these tissues before long and starch grains can be observed in about 3-week old cultures, indicating that they are metabolically active. Thus, they can transport and store nutrients, much of which can reach the developing microsporeso The young c a l l i liberated from the spore wall contain a high concentration of amorphous l i p i d s and a moderate level of starch grains. At a l a t e r stage, they accumulate more of these substances. Cell d i f f e r e n t i a t i o n then takes place within each callus. Some peripheral c e l l s became the primordia of l e a f l i k e protrusions and embryoids. Since our observations were made from the materials collected within 40 days a f t e r the culture i n i t i a t i o n , no well organized embryoids were observed in our c a l l i . Genovesi and Magill (1982) reported that both organogenesis and embryogenesis can take place in rice c a l l i d e r i ved from microspores. The presence of l e a f - l i k e protrusions and globular structures at the surface of our c a l l i are in agreement w~th t h e i r findings. One i n t e r e s t i n g finding in our experiments should be mentioned here. At about the lO0-cell stage, each m u l t i c e l l u l a r microspore consists of two types of c e l l s , a large and small class. This d i f f e r e n t i a t i o n of c e l l s was observed with a l l three staining procedures. The large c e l l s are rich in amorphous l i p i d s and starch grains but stain l i g h t l y withTBO while the small ones have much less l i p i d and no starch grains but stained with TBO much more intensely. Thus, they seem to perform quite d i f f e r e n t functions, the former probably n u t r i t i v e and the l a t e r reproductive. A more detailed study of the d i f f e r e n t i a t i o n of these
two types of c e l l s , especially t h e i r ontogenetic relationship with the vegetative and generative c e l l , and t h e i r roles in the developing callus should be carried out before a d e f i n i t e conclusion can be reached. ACKNOWLEDGEMENTS We thank the National Science Council f o r f i nancial support. REFERENCES Bajaj YPS (1983) In:Evans, Sharp, Ammirato and Yamada (eds.), Handbook'-of Plant Cell Culture. Macmillan Pub. Co., New York. pp. 228-287. Bourgin JP, Nitsch JP (1967) Ann. Physiol. Veg. 9: 377-382. Chao CY (1977) Amer. J. Bot. 64:921-930. Chao CY (1979) Phytomorphology 29:381-387. Chayer J, Bitensky L, Butcher RG (1973) Practical Histochemistry. Wiley, London. Chen CC (1977) In Vitro 13:484-489. Chen CC, Wu YH (1983) Proc. Natl. Sci. Counc. Bo, ROC 7:151-157o Chen LJ, Lai PC, Liao CH, Tsay HS (1982) J. Agric. Res. China 31:283-290. Chu CC, Wang CC, Sun CS, Chen H, Yin KC, Chu CY, Bi FY (1975) Sin Sinica 18:659-668. Esau K (1977) John Wiley and Sons, New York. Genovesi AD, Magill CW (1982) Plant Cell Report I : 257-260. Guha S, Maheshwari SC (1964) Nature 204:497. Guha-Makherjie S (1973) J. Exp. Bot. 24:139-144. l y e r RD, Raina SK (1972) Planta 104:146-156. Murashige T, Skoog F (1962) Physiol. Plant. 15:473497. Niizeki H, Oono K (1968) Proc. Jap. Acad. 44:554-557. Pearse AGE (1968) Histochemistry, Theoretical and Applied. Vol. I . J. and A. Churchill, Ltd., London. Raghavan V (1976) Science 191:188-189. Raghavan V (1979a) Amer.J. Bot. 66:36-39. Raghaven V (1979b) Amer. J. Bot. 66:784-795. Raghaven V (1981) J. Cell Biol. 89:593-606. Sangwan-Norrell BS (1978) Can. J. Bot. 56:805-817. Sunderland N, Collins GB, Dunwell JM (1974) Planta 117:227-241. Sunderland N, Wicks FN (1971) J. Exp. Bot. 22:213-216.
