Morphological studies in the Gramineae

M O R P H O L O G I C A L S T U D I E S IN T H E G R A M i N E A E

IV. Embryology of Eleusine indica Gaertn. and Dactyloctenium aegyptium (Desf) Beauv.*

BY NARESH CHANDRA (School of Plant Morphology, Meerut)

Received April 20, 1963

(Communicated by Dr. V. Purl, F.A.SC.)

INTRODUCTION

EMBRYOLOGICAL studies in the Gramineae have been confined mostly to the cereals and certain forage grasses (see Artschwager et aL, 1929; Randolph, 1936; Juliano and Aldama, 1937; Cooper, 1937; Merry, 1941; Khosla, 1946; Artschwager and McGuire, 1949; Narayanaswami, 1952-55, etc.). A study of certain locally occurring wild grasses was undertaken to elucidate details of embryological features including post-fertilization changes leading to fruit formation. The present communication deals with the embryology and fruit development of Eleusine #Tdica and Dactyloctenium aegyptium both belonging to the tribe Eragrosteae of the subfamily Pooideae. Earlier observations on E. indica relate to the development of integument and germination (Cummins, 1929).. Narayanaswami (1952, 1955) has worked out in detail the developmental morphology of the caryopsis of E. coracana the finger millet.

MATERIAL AND METHODS

Spikes at various stages of development were fixed in F.A.A. Outer glumes of the spikelets were removed before dehydration. Customary methods of dehydration, clearing and embedding were followed. Sections were cut at 8--16~ thickness and stained with Heidenhain's iron-alum haematoxylin and safranin-fast green.

OBSERVATIONS

Both the species show a close resemblance in the mode of development and hence only Eleusine indica will be described in detail and reference will be made to D. aegyptium wherever necessary.

* Research contribution from the School of Plant Morphology, Meerut Coll~e, Meerut.

BI 117

118 NARESH CHANDRA

Microsporogenesis and the male gametophyte.--Each anther contains four microsporangia in the usual manner. Their wall is four-layered, a thin- walled epidermis followed by an endotheciat layer, a middle layer and a tapetum which is glandular in nature (Fig. 1). The tapetal cells in both the species become binucleate with the two nuclei lying appressed to each other (Fig. 1). In a few grasses, however, tapetal cells remain uninucleate throughout, e.g., Pennisetum typhoides (Narayanaswami, 1953)and Panicum miliare ('Narayanaswami, 1955). In young anthers the pollen mother cells are very compactly arranged in one or two rows and their number varies from eight to ten per microsporangium. The middle layer shows signs of degeneration at a time when pollen mother cells enter into the first division of meiosis or even earlier (Fig. 1)~ The pollen mother cells separate off frcm one anclher, enlarge in size and attain an ellipsoidal configuration before they commence to divide. Meiosis is normal and microsporegenesis is of the successive type. An isobilateral tetrad of microspores is produced (Figs. 2-4). It has been noticed in D. aegyptium that the divisions in the two dyad cells may not synchronize. The tapetum is almost completely absorbed during the course of microsporogenesis. The middle layer also degenerates but its relics can be easily made out in the form of very thin dark streaks below the endothecial layer. The cells of the endothecial layer develop characterristic fibrous thickenings.

The microspores round off and enlarge in size (Fig. 5). Later on a -mall lens-shalzed generative cell is cut off on one side (Fig. 6). The generative cell gives rise to two male cells which are to begin with spherical but later on become slightly curved spindle-shaped bodies lying close to the tube nucleus (Fig. 7). The tube nucleus shows signs of degeneration in the pollen grain itself and stains deep black with Heidenhain's haematoxylin-- a condition also observed in the millets (Narayanaswami, 1953-55). The pollen grain has a smooth exine with a single pore and is three-celled at the time of shedding. The only wall layers persisting at the time of dehiscence are the epidermis and the endothecial layer (Fig. 8).

The ovule.--The ovary as usual in the Gramineae contains a single ovule attached to its posterior wall. This at the time of inception is a small pro- tuberance. As the integuments develop the ovule begins to curve and finally becomes semi-anatropous. The ovule is bitegmic and tenuinucellate (Fig. 18). Both the outer and the inner integuments are two-layered. The inner integument, however, may be three-layered in the micropylar region. The micropyle is formed by the inner integument alone and the outer integument remains slightly below the micropylar region (Fig. 18).

Morphological Studies in the Gramineae--IV 119

Megasporogenesis and the female gametophyte.--A single hypodermal archesporial cell differentiates in the nucellus even before the initiation of integuments. This archesporial cell does not cut off any parietal cell and directly behaves as the megaspore mother cell (Figs. 9, 10). The megaspore mother cell increases in size and undergoes meiosis to form a linear tetrad

l,® 1

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10

12

11

of megaspores (Fig. 11). D. aegyptium (Fig. 12).

,)

FIGS. l- 17

A T-shaped

0 14

14

tetrad may also be. formed as in The micropylar three megaspores degenerate while

the chalazal one functions. The functional megaspore increases in size to a great extent and has a prominent nucleus situated centrally (Fig. 11).

The nucleus of the functional megaspore divides to form two nuclei which soon migrate to opposite poles. By two more successive divisions

120 NARESH CHANDRA

in these two nuclei an eight-nucleate embryo-sac is formed (Figs. 13-16). One nucleus from the chalazal side migrates towards the micropylar side and pairs with one from this side to form the polars (Figs. 16 and 17). Meanwhile the micropylar three nuclei organize into the egg-apparatus and the chalazal three into the antipodals (Fig. 17). In D. acgyptium the antipodals subsequently divide to give rise to 6-10 cells which enlarge in size to a great extent and present the most conspicuous feature of the female gametophyte (Fig. 21). In E. hMica on the other hand the antipodals do not increase in number (Fig. 23). Multiplication of the antipodal cells is a very common feature in the Gramincae (see Shadowsky, 1926; Randolph, 1936; Narayanaswami, 1953-55, etc.). Antipodals are uninucleate in D. aegyptium and E. indica and do not become coenocytic as has been observed in Cynodon dactylon, Agrostis canina, etc. (Shadowsky, 1926) and Pennisetum typhoideum ; Paspalum scrobiculatum and EchinochIoa frumentacea (Narayanaswami, 1953, 1954 and 1955 respectively). In Pennisetum typhoideum as many as 3-8 nuclei have been recorded in each antipodal cell which finally coalesce together before degeneration. Another feature in Eleusine indica is the varia- tion in shape exhibited by the antipodals which are sometimes balloon-like. The two polars lie appressed to each other near the egg apparatus but do not fuse until after fertilization. The development of the embryo-sac thus follows the Polygom, n-type. The synergids have prominent beaks and the nuclei lie in their upper halves (Figs. 19 and 20). The egg is bigger in size and is uniformly vacuolated with the egg nucleus lying in the centre or slightly towards the lower side.

The embryo-sac at maturity is somewhat curved. The antipodals in both the species come to occupy a lateral position in the embryo-sac toward the point of attachment of the ovule (Figs. 21 and 23). On the other hand in some panicoid grasses it has been observed that they retain their chalazal position until the time of degeneration (Randolph, 1936; Narayanaswami, 1953-55).

Fertilization.--The actual course of the pollen tube was not traced. One synergid may or may not be destroyed by the pollen tube. One of the male gametes (d'l) which is a very small, spherical body was found lying appressed to the egg nucleus and the second male gamete (if2) was found lying in close proximity to the polars (Figs. 21 and 22). These stages indicate that double fertilization, i.e., syngamy and triple fusion takes place.

Embryogeny.--The zygote after a period of rest undergoes a transverse division forming a small terminal ceil and a bigger much vacuolated basal


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122 NAKESH CHANDRA

cell (Fig. 26). The basal cell divides first transversely so that a three- celled proembryo is produced (Figs. 27 and 32). The terminal cell now divides vertically giving rise to the 4-celled condition (Figs. 28 and 33). The subterminal cell divides vertically in D. aegyptium (Figs. 34 and 35) but in E. indica in one case it was seen to divide transversely (Fig. 29). By further vertical and transverse divisions in the cells of the terminal as well as subterminal tier a globular mass of cells is produced (Fig. 30). The basal cell by a transverse division gives rise to two cells which again divide transversely and form the suspensor (Figs. 26-31). At the time of commencement of organ differentiation the embryo is a globular mass of cells with a 4-6-celled suspensor. Further development of the embryo conforms to the pattern observed in other Gramineae.

Endosperm.--The endosperm is of the nuclear type. Soon after triple fusion the product undergoes free nuclear divisions which have a marked tendency to occur simultaneously specially in the earlier stages of development (Figs. 23-25). After a number of free nuclear divisions have taken place wall formation starts. Walls are first laid down around the nuclei lying in close vicinity of the developing proembryo (Fig. 25). Wall formation now proceeds in the chalazal portion also. In the later stages of endosperm development cell divisions remain confined to the peripheral layer (Fig. 40). Cells thus cut off on the inner side contribute to the endosperm tissue while the peripheral layer after the cessation of activity becomes the aleurone layer of the mature seed. Such a behaviour was first observed by Gordon (1922) and has been subsequently confirmed by several authors. The endosperm is full of starch-filled cells. It has been noticed that the cells of the endosperm abutting the scutellum either contain very little starch or are completely empty.

Fate of nucellus, integuments and ovary walL--The antipodals take up a lateral position in the embryo-sac where they degenerate after some time. Weatherwax (1926)and Randolph (1936), however, observed the persistence of antipodal cells at the chalazal end of the endosperm in a mature caryopsis of Zea mays. Doubts have been cast on such observations by Narayanaswami (1954) who is of the opinion that such proliferated antipodal cells as have been described by these authors to persist in a mature caryopsis are actually some endosperm cells involuted in, rather than antipodal cells. Such a condition has been observed by him in Paspalum scrobieulatum (1955) and by Sass (1946) in an argentine variety of waxy maize. The whole of the nucellus but for the nueeUar epidermis is also absorbed (Fig. 37). The nucellar epidermis also degenerates at a later stage (Figs. 38 and 39). In


some grasses, however, the nucellar epidermis persists as a definite layer ~ the perisperm, e.g., Oryza sativa (Juliano and Aldama, 1937), Eragroatis cilianencis (Stover, 1927); Poa pratensis (Anderson, 1927), Zea maya (Randolph, 1936), Euchlaena mexicana (Cooper, 1937) and several millets (Narayanaswami, 1953-55). Out of the two integuments the outer one degenerates early after fertilization (Figs. 37-39). On the other hand cells

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of both the layers of inner integument enlarge in size to a great extent (Figs. 37-39). Particularly the cells of the inner layer undergo rapid increase in thickness so much so that they become two or three times their original thickness. With the increase in thickness, there is an increased impreg- nation of some brownish material in these ceils (Fig. 39). The outer layer gets thickened first on the outer tangential walls but soon it becomes a con- tinuous thickened layer (Fig. 40). Thus the seedcoat is derived from the two layers of the inner integument. The present observations on development of integument are in full accord with those of Cummins (1929) on Eleusine indica. Thus we find that in these species there is present a true seedcoat derived from the two layers of the inner integument. In Zea mays (Randolph, 1936) and several other grasses, however, both the integuments degenerate completely and it is the pericarp which becomes very hard.

124 NARESI-I CHANDRA

The ovary wall is five to eight cells thick to begin with but soon the cells of the middle layers disintegrate (Figs. 37-39). At maturity the pericarp becomes a very thin loose papery membrane round the seedcoat and it does not go into fusion with the seedcoat. The fruit is thus an utricle (Fig. 41).

The fruit.--A major portion of the mature fruit is occupied by the endosperm which is full of starch grains except for the outermost aleurone layer (Fig. 40). The embryo has a massive scutellum'which abuts directly with the endosperm (Fig. 41). The cells of the outermost layer of the scutellum are comparatively smaller but do not constitute a very well-defined epithelial layer (Fig. 42). Opposite the scutellum there is an outgrowth, the epiblast that is directed towards the coleoptile.

SUMMARY

The present paper deals with the embryology and fruit development of two wild grasses, Eleusine indica and Dactyloctenium aegyptium, both belonging to the tribe Eragrosteae of the subfamily Pooideae.

Microsporogenesis is o f the successive type and results in isobilateral tetrads of microspores. The microspores round up and a generative cell is cut off which later gives rise to two male cells which are spindle-shaped. The pollen grain at the time of shedding is three-celled and has a smooth exine with a single pore. The tube nucleus begins to degenerate while still in the pollen grain.

The ovule is semi-anatropous, bitegmic and tenuinucellate. A single hyFoderrnal archesporial cell differentiates in the nucellus which behaves directly as the megaspore mother cell. It undergoes meiosis to form a linear or a T-shaped tetrad of megaspores. The chalazal megaspore is functional and the development of embryo-sac conforms to the Polygonum- tyl:e. The antipodals become very large and divide in D. aegyptium while in E. indica the primary number of three is retained. In any case they come to occupy a lateral position in the ovule. Double fertilization takes place.

The development of the embryo conforms to the type met with in other grasses. The endosperm is of the nuclear type but afterwards becomes cellular and its peripheral layer behaves as a cambium which cuts of cells on the inner side. On the cessation of activity it becomes the aleurone layer of the seed.

The outer integument and the nucellus are completely absorbed in the development of fruit and it is the inner integument which forms the seed-


coat. The middle layers of the pericarp collapse and at maturity it is in the form of a loose covering round the seed.

The fruit is an utricle. The major part of the seed is occupied by the endosperm which is full of starch. The mature embryo has a scutellum that abuts with the endosperm. Opposite the scutellum there is the epiblast. The stem tip is covered by a few leaf primordia and coleoptile while the radicle is protected by a root-cap and the coleorhiza.

ACKNOWLEDGEMENTS

G r a t e f u l t h a n k s a r e d u e to P r o f e s s o r V. Pur i , D.SC., F.N.I., f o r g u i d a n c e a n d e n c o u r a g e m e n t a n d to D r . Y . S. M u r t y fo r h e l p f u l sugge s t i ons . T h a n k s a r e a l so d u e to P r o f e s s o r P. M a h e s h w a r i f o r t he l o a n o f s o m e l i t e r a t u r e .

Anderson, A. M.

Artschwager, E., Brandes, E. W. and Starrett, R. C.

and McGuire, R. C.

Cooper, D. C.

Cummins, M. P.

Gordon, M. ..

Juliano, J. B. and Aldama, M. S.

Khosla, S. ..

Merry, J. ..

Narayanaswami, S.

REFERENCES

.. "Development of the female gametophyte and caryopsis of Poa pratensis and Poa compressa," Jour. Agr. Rex., 1927, 34, 1001-18.

"Development of flower and seed of some varieties of sugar- cane," Ibid., 1929, 39, 1-30.

"Cytology of reproduction in Sorghum vulgate," Ibid., 1949, 78, 659-73.

.. "Macrosporogenesis and embryo-sac development in Euch- laena mexicana and Zea mays," Ibid., 1937, 55, 539-51.

.. "Development of the integument and germination of the seed of Eleusine indica," Bull. Tort. Bot. Club, 1929, 56, 155-62.

"The development of endosperm in cereals," Proc. Roy. Soc. Victoria, 1922, 34, 105-16.

"Morphology of Oryza sativa L.," Philippine J. Agric., 1937, 26, 1-134.

"Developmental morphology in some Indian millets," Proc. lnd. Acad. Sci., 1946, 24B, 207-24.

"Studies on the embryo of Hordeum sativum, I. The development of the embryo," Bull. Torr. Bot. Club, 1941, 68, 585-98.

.. "Microsporogenesis and male gametophyte in Eleusine coraeana Gaertn.," Curt. Sci., India, 1952, 21, 19-21.

.. "The structure and development of the caryopsis in s o m e Indian millets. I. Pennisetum typhoideum Lich.," Phyto- morphology, 1953, 3, 98-112.

. . "The structure and development of the caryopsis in some Indian millets. III. Paspalum serobiculatum L. Bull. Torr. Bot. Club, 1954, 81~ 288-99.

126

Narayanaswami, S.

Randolph, L. F.

Sass, J. E.

Shadowsky, A. E.

Stover, E. L.

Weatherwax

NARESH CHANDRA

.. "The structure and development of the caryopsis in some Indian millets. II. Panicum miliare Lamk. and P. miliaceum Linn.," Lloydia, 1955, 18, 61-73.

.. "The structure and development of the caryopsis in some Indian millets, IV. Echinochloa frumentacea Link.," Phytoraorphology, 1955, 5, 161-71.

.. "The structure and development of the caryopsis in some Indian millets. V. Eleusine coraeana Gaertn," Papers Michigan Acad. Sciences, Arts and Letters, 1955, 40, 33-46.

.. "Developmental morphology of the caryopsis in Maize," Jour. Agr. Res., 1936, 53, 881-916.

.. "The development of endosperm and antipodal tissue in Argentine waxy maize," Am. Jour. Bot., 1946, 33, 791-95.

.. "Der antipodiale Apparat bei Gramineen," Flora, 1926, 120, 344-70.

.. "The embryo-sac of Eragrostis cilianensi~ (All) Link. A new type of embryo-sac and a summary of grass embryo-sac investigation," Ohio .four. Sci., 1927, 37, 172-81.

.. "Persistence of the antipodal tissue in the development of the seed of maize," Bull. Torr. Bot. Club, 1926, 53, 381-84.

EXPLAN.~TION OF FIGURES

FIGS. 1-17. (Figs. 1-11 and 13-17. Eleusine indica. Fig. 12. Dactyloctenium aegyptium). Fig. 1. L.s. portion of an anther lobe showing the wall layers and the microspore mother cells, x 285. Figs. 2-7. Different stages of microsporogenesis and male gametophyte, × 285. Fig. 8. L.s. portion of a mature anther showing the epidermis and the endothecial layer with fibrous thickenings, × 285. Fig. 9. A young ovule showing the hypodermal megaspore mother cell, x 285. Fig. 10. Meiosis I in the megaspore mother cell, × 285. Fig. 11. Linear tetrad of megaspores, × 285. Fig. 12. A T-shaped tetrad of megaspores in D. aegyptium, x 182. Figs. 13-17. Different stages in the development of embryo-sac, x 285.

F/QS. 18-36. (Figs. 18, 19, 22-31. Eleusine indica. Figs. 20, 21, 32-36. Dactyloctenium aegyptium.) Fig. 18. L.s. ovule, x g0. Fig. 19. Egg apparatus and polar nuclei, x 200. Fig. 20. Egg apparatus in D. aegyptium, x 250. Fig. 21. Mature embryo-sac of D. aegyptium just after fertilization, x 112. Fig. 22. Upper portion of the embryo-sac showing syngamy and triple fusion, × 200. Fig. 23. Embryo-sac showing the product of division of the primary endosperm nucleus, degenerating antipodais, degenerating synergids and the zygote, x 120. Fig. 24. Mieropylar portion of the ovule showing the free nuclear endosperm and a three- celled procmbryo, x 90. Fig. 25. Early stage of endosperm formation. Note the walls have been laid down in close vicinity of the proembryo, x 90. Figs. 26-31. Earlier stages of develop. merit of embryo, x 200. Figs. 32-36. Earlier stages of development of embryo in D. aegypo ium, 250. (~nt, antipodals; emb, embryo; end, endosperm; ii, inner integument; mic, micropyca; he, nucdlar epidermis; nu, nueellus; oi, outer integument; sy, synergids.)


FIGs. 37-42. Eleusine indica. Figs. 37-40. Peripheral portions of the developing fruit in L.s. showing collapse of outer integument, nucellus and the middle layers of pericarp and the development of seedcoat from inner integument, pericarp not shown in Fig. 40, x 250. Fig. 41. L.s. lower portion of the fruit showing the different parts of the mature embryo, the endosperm, seedcoat and loose pericarp, x 112. Fig. 42. Portion of the scutellum showing epithelial layer, x250. (al, aleurone layer; or, coleorhiza; coleoptile; epi, epiblast; epl, epithalial layer; 1, leaf primordia; pc, pericarp; rc, root cap; s, scutellum; s, stem tip; sc, scutellum.)

Morphological studies in the Gramineae

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