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Embryogenesis in Najas marina L.: Structural and histochemical approach
Aquatic Botany, 22 ( ! 9 8 5 ) 45--60
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Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
EMBRYOGENESIS HISTOCHEMICAL
IN NAJA8 MARINA L.: STRUCTURAL APPROACH
AND
M.R. V I J A Y A R A G H A V A N and TRIPAT KAPOOR
Department of Botany, University of Delhi, Delhi-110007 (India) (Accepted 15 March 1985)
ABSTRACT
Vijayaraghavan, M.R. and Kapoor, T., 1985. Embryogenesis in Najas marina L.: Structural and hlstochemicalapproach. Aquat. Bot., 22: 45--60. In Najas marina L. few polysaccharide grains are observed in zygote, basal cell and embryonal cells until the initiation of embryonic shoot-apex. With the formation o f the shoot-apex, numerous polysaccharide grains engorge in the embryonal cells. The basal cell wall, subjacent to the nucellus, stains intensely with PAS (Periodic Schiff's)-reaction. The concentration of proteins and R N A increases in the basal cell. Interestingly, the e m b r y o shows intraseminal germination. The cells o f embryonic shoot-apex, embryonic leaves, r o o t primordium and procambial cells show a few polysaccharide grains while the cells of h y p o c o t y l e d o n a r y and cotyledonary regions are engorged with polysaccharide (starch) grains. Uniform distribution o f proteins and RNA is observed in the embryonic shoot-apex, embryonic-leaves, root primordium and procambium, but the cells of h y p o c o t y l e d o n a r y and c o t y l e d o n a r y zones exhibit a low profile for these metabolites. The initial root-primordium remains quiescent. Three or 4 epidermal cells, subjacent to this quiescent primordium, differentiate; show densely stained, polarised, protein bands; and act as the future r o o t primordium. The nucleus of the basal cell becomes p o l y p l o i d and densely stains for proteins, RNA and DNA. At the globular p r o e m b r y o stage, numerous nucleolar bodies migrate towards the periphery of the nucleus and at the 3-leaf e m b r y o stage, these nucleolar bodies, rich in proteins and RNA, are located in the cytoplasm revealing nucleo-cytoplasmic interaction. The basal cell that never divides, but only enlarges, is persistent in the mature seed.
INTRODUCTION The information on structural development of aquatic monocotyledonous embryogenesis is meagre (Maheshwari, 1950). Little work exists on the cytochemical aspects of aquatic angiosperm embryogenesis. Therefore, the present work on Najas marina L. was undertaken to study the distribution pattern of various metabolites during embryogenesis and intraseminal germination.
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© 1985 Elsevier Science Publishers B.V.
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MATERIAL AND METHODS Post-fertilized flowers and fruits (at various stages of development) of Naias Marina were fixed in FAA (Formalin Acetic Acid) and AA (Acetic Alcohol). The fixed material was dehydrated in a tertiary-butyl-alcohol series and embedded in paraffin wax. Twelve gm sections were cut and mounted on chemically-cleaned slides using Haupt's adhesive. The material that was fixed in FAA was used for the localization of insoluble polysaccharides and proteins, and that fixed in AA was used for nucleic acids. Insoluble polysaccharides were localized with the PAS (Periodic Schiff's) technique (Jensen, 1962), proteins with the mercuric-bromophenol blue method (Mazia et al., 1953), RNA by the Pyronin-Y method (Tepper and Gifford, 1962) and DNA by the Feulgen reaction (Kallarackal, 1974). Control slides were made in order to check the specificity of the various reactions. Starch grains were confirmed by IKI (Potassium Iodide) test. RESULTS
Structural aspects Najas marina (Fig. 1A) is a dioecious plant. The female flower is devoid of a perianth. The gynoecium is unilocular and uniovulate. The style and stigma are conspicuous in the fruit (Fig. 1B). The zygote (Fig. 2A) divides transversely resulting in a large basal cell and a small terminal cell. The basal cell remains undivided and functions directly as the embryo suspensor cell whereas the terminal cell undergoes numerous mitotic divisions and forms the organogenetic part of the embryo. Further growth and differentiation in this part of the embryo result in globular (Fig. 3A) and mature embryos. The embryo development follows the conventional Caryophyllad type (Maheshwari, 1950). Intraseminal germination The embryo does not undergo a dormant phase/after-ripening period and germinates precociously to produce a well developed embryonic shootapex (Figs. 2F, 4C) and upto 3 leaves (Fig. 2E) within the seed. The embryo thus reveals the following features: (1) basal cell; (2) root primordium; Fig. 1A--H. Na]as marina, external morphology (A and B), localization of insoluble polysaccharides (C--H); (A) Vegetative portion of female plant X 6; (B) A fruit showing persistent stigma (s) x 12; (C,D,G) Portions of embryo at 3 leaf stage showing root primordium (r) with basal cell (bc), embryonic shoot-apex (pv) and cotyledonary tip (c), respectively: C × 200; D × 450; G X 390; (E) Three epidermal cells (arrows) that differentiate are the new root initials. The polysaccharide grains cluster around nuclei (nu) in these cells x 390; (F,H) A few cells from hypocotyledonary and cotyledonary regions. The epidermal cells (ed) in H show polarized distribution of polysaccharide grains F X450, H X 390.
47
A
,~. 2A--F. Najas marina, localization of total proteins (A,C--E) and RNA (B,F); (A) gote (z), persistent synergid (sy) and nucellar cells (n) are protein rich × 270; (B) .~globular embryo showing basal cell (bc) and organogenetic portion rich in RNA 270; (C,D) Preglobular and globular embryos stained densely for proteins. Inset in shows the basal cell at globular embryo stage × 200; (E) Portion of embryo showing bryonic shoot-apex (pv) and 3 embryonic leaves (1) × 135; (F) Portion of seed showing raseminal germination. The cells of root primordium (r), procambium (pl), embryonic )ot-apex and embryonic leaf stain uniformly for RNA and cells of hypocotyledonary ;), cotyledonary (c) zones and persistent nucellus (pr) are poor in cytoplasmic RNA. e degenerating chalazal endosperm chamber (cc) persists × 100.
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B
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Fig. 3A--H. Najas marina, localization of DNA; A--D. Basal cell (bc) and embryonal cells during progressive advancing stages of embryogeny × 200; (E) Portion of mature embryo, and a few cells of procambium (pl) x 390; (F) Cells of embryonic shoot-apex (pv) and embryonic leaf (1) with DNA-rieh nuclei x 390; (G) Portion of cotyledon to show that hila (arrows) are feulgen-positive. Binary fission of the hilum is noteworthy x 850; (H) Portion of e m b r y o at 3-leaf stage to show that the intensity of DNA staining is reduced in the nuclei of cotyledonary cells (c) x 200.
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I
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(3) hypocotyledonary zone; (4) cotyledonary region; (5) procambium; (6) embryonic shoot-apex; (7) embryonic leaves. The cells of root primordium (Fig. 2F), procambium (Fig. 2F), embryonic shoot-apex (Fig. 2F) and embryonic leaves (Fig. 2E) are small and have uniform distribution of cytoplasm whereas the cells of hypocotyledonary and cotyledonary (Fig. 2F) regions are large and vacuolate with thin cytoplasmic contents. Embryo basal cell
The basal cell develops into a vesicular structure (Fig. 2D inset). The nucleus becomes polyploid. At about the 3-1ear embryo stage, prominent nucleolar-blebs (Fig. 4G, arrows) surrounded by hyaline areas are seen in the cytoplasm. The basal cell is persistent (Figs. 1C, 4B) in the mature seed and perhaps prevents the emergence of the original root-primordium from the radicular end. Therefore, the new root-primordium (Fig. 4D) differentiates from the epidermal cells located toward the lateral sides of the original root-primordium. Testa and pericarp
At zygote and preglobular embryo stages, the inner integument is 2layered except at the micropylar end where it is 3-layered (Fig. 5A). During later progressive advancing stages of embryo development, these 2 layers, except at the micropylar end, become compressed. The outer integument, at the zygote stage, comprises 4 or 5 layers of cells. The cells of the outer epidermis expand radially and tangentially, whereas the other layers increase in number (Fig. 5B). At the 3-embryonic leaf stage, 7 layers of cells sandwiched between the 2 epidermes develop sclerenchymatous cell wall thickenings and pittings (Figs. 5C--E). The pericarp (Fig. 5B) except at the chalazal end, remains thin and 2-layered and shows little morphological or histological changes throughout the embryogenesis.
Fig. 4A--H. Najas marina, localization o f total proteins ( A - - F ) and R N A (G,H); (A,B) Portions of embryos to show cells o f r o o t primordium (r) that stain uniformly for cytoplasmic and nuclear proteins. The basal cell (bc) is rich both in cytoplasmic and nuclear proteins A X 100, B × 400; (C) Cells o f embryonic shoot-apex (pv) and embryonic leaves (l) showing homogeneous distribution of proteins X 400; (D) A few epidermal cells of the h y p o c o t y l e d o n a r y region that show polarised protein bands (arrows), are the new root primordium initials × 400; (E) Portion of the cotyledonary cells (c) × 400; (F) A few h y p o c o t y l e d o n a r y cells showing procambium (pl) x 400; (G) Basal cell at 3-leaf embryo stage to show pyroninophilic nucleolar-blebs (arrows) and nucleolus X 900; (H) A few cotyledonary cells showing congregation of starch grains around the nuclei (nu), hila (arrows) are pyroninophilic x 900.
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3 Fig. 5A--E. Najas marina, localization of insoluble polysaccharides; (A) Testa at preglobular embryo stage. Inner integument (ii) is 3-layered at micropylar region × 200; (B) Longisection of fruit at embryo (em) stage. The pericarp (pc) is 2-layered x 80; (C) Portion of longisection of fruit at 3-leaf embryo stage x 200; (D,E) A few cells from the outer integument (oi) at 3-leaf embryo stage showing secondary cell wall thickenings (arrows), except epidermes (ed), pittings and polysaccharide grains (ps). In E, pittings are seen in surface view x 400.
Histochemical aspects Insoluble polysaccharides The z y g o t e , preglobular- and g l o b u l a r - e m b r y o s s h o w small p o l y s a c c h a r i d e grains. W h e n t h e e m b r y o n i c s h o o t - a p e x is well d e v e l o p e d , small p o l y s a c charide grains are localized in h y p o c o t y l e d o n a r y a n d c o t y l e d o n a r y cells,
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but the cells of the root primordium, procambium and embryonic shootapex are devoid of such grains. The root primordium, procambium and embryonic shoot~pex, at the l-leaf embryo stage, have few polysaccharide grains, whereas the cells of hypocotyledonary and the cotyledonary regions are engorged with this metabolite. However, at the 3-leaf embryo stage root primordium (Fig. 1C), embryonic shoot-apex (Fig. 1D), procambium and embryonicleaves reveal small polysaccharide grains while the cells of hypocotyledonary (Fig. 1F) and cotyledonary (Fig. 1G) regions have copious deposition of starch grains. The alignment of such polysaccharide grains in various regions of the embryo is interesting. In root primordium (Fig..1C), procambium and embryonic shoot-apex (Fig. 1D) small polysaccharide grains are seen at random. In hypocotyledonary (Fig. 1F) and cotyledonary (Fig. 1G) regions, the polysaccharide grains (starch) congregate around the nucleus. In the epidermal cells such grains show polarized distribution (Fig. 1H) and are restricted to the abaxial side. Three or 4 epidermal cells, adjacent to the hypocotyledonary r e g i o n a n d below the root primordium, differentiate as the new future root primordium initials, and contain polysaccharide grains compactly clustered around the nucleus in each cell (Fig. 1E). The embryo basal cell (Fig. 1C) forms a cap-like structure over the radicular end, and throughout embryogenesis shows an intense PAS-positive cell wall, but lacks polysaccharide grains. During progressive advancing stages of embryogenesis, few polysaccharide grains are localized in the nucellus. At the 3-leaf embryo stage, the nucellar cells that persist at the micropylar, chalazal and the lateral sides flanking the embryo constitute the perisperm and are faintly stained. The cells of the inner integument (Fig. 5A), at preglobular and globular embryo stages, are devoid of polysaccharide grains. The cells of the outer integument, except the outer and the inner epidermes, reveal polysaccharide grains and at about the mature embryo stage, the polysaccharide contents increase enormously (Fig. 5B). At the 3-leaf embryo stage, prominent, PAS-positive, cell-wall thickenings (Figs. 5C--E) and abundant polysaccharide grains (Figs. 5D,E) are present in all layers of the outer integument except the epidermes (Fig. 5C). The cell walls of the pericarp are PASpositive (Fig. 5B), but the cells lack polysaccharide grains during embryogenesis. Proteins and nucleic acids
The zygote (Fig. 2A) is rich in proteins and RNA. Further, at octant, globular (Figs. 2B--D) and mature embryo stages, the basal cell and the embryonal cells are protein and RNA rich. The zygote nucleus stains feebly for DNA. At the 2-ceUed pro-embryo stage, the nucleus of the terminal cell stains with lesser intensity than that of the basal cell. The nuclei of embryonal cells at globular (Fig. 3A) and mature (Fig. 3B) embryo stages are rich in DNA content. The basal cell nucleus, during embryogenesis,
;ains densely for DNA (Figs. 3A--D) when compared to the embryonal ells. At 1--3-leaf embryo stages, cells of root-primordium (Fig. 4A), embryonic ~oot-apex (Fig. 4C), embryonic leaves (Fig. 2E) and procambium (Fig. F) stain uniformly for proteins (Figs. 4A,C,F) and RNA (Fig. 2F). The ells of the hypocotyledonary (Fig. 4F) and the cotyledonary (Fig. 4E) reions have dense nuclear proteins (Figs. 4E,F) and RNA (Fig. 2F), but the ytoplasmic proteins and RNA are less. The proteins in these cells are fibrillar i nature (Fig. 4F). Three or four epidermal cells (arrows) subjacent the riginal root-primordium differentiate, show densely-stained and polarized rotein bands (Fig. 4D) and act as the new initials of the future root priLordium. At the l-leaf embryo stage, nuclei of root primordium (Fig. C), procambium (Fig. 3E), embryonic shoot-apex and leaf (Fig. 3F), ; well as the embryonal cells stain for DNA. The nuclei of hypocoty~donary and cotyledonary (Fig. 3H) cells, at 3-leaf embryo stage, show ~ss intensity for DNA. The starch grains present in these cells have Feulgenositive (Fig. 3G) and pyroninophilic hila (Fig. 4H). Hila of such grains ~hibit binary fission (Fig. 3G). The basal cell at this stage is partially Jtolytic, but persistent and reveals 3--6 nucleolar blebs which axe pro~,in-rich and pyroninophilic (Fig. 4G). The nucellar cells at zygote (Fig. 5A), preglobular (Fig. 2C) and globular nbryo stages, are protein and RNA rich, whereas at the mature embryo age, the intensity of the protein and the RNA contents is very low. owever, the cells of the 3-layered nucellus that persist at the micropylar ~gion stain for nuclear proteins and nucleolar RNA, but those cells that ank the embryo are poor both in protein and RNA (Fig. 2F) contents ld even their nuclei stain feebly for DNA. The cells of integuments and ~ricarp reveal a decreasing trend of protein and RNA contents during nbryogenesis. [SCUSSION ygote and embryo In Najas marina the polysaccharide grains are absent around the zygote Jcleus. This is in contrast to the accumulation of polysaccharide grains ,en around the zygote nucleus in Gossypium hirsutum L. (Jensen, 1968), zpsella bursa-pastoris (L.) Medic. (Schulz and Jensen, 1968a), Iberis amara . (Prabhakar, 1979) and Linum usitatissimum L. (Bhat and Vijayaraghavan, ~80). At the 2~elled, preglobular and globular proembryo stages no polyccharide grains are localized in the basal cell and embryonal cells of Najas arina. In Capsella bursa-pastoris (Schulz and Jensen, 1968a), at the 2~lled stage, polysaccharide grains are more in the basal cell than in the rminal cell, but in Lilium spp. (Georgieva, 1965) and Ranunculus sceletus L. (Vijayaraghavan and Bhat, 1982) more polysaccharide grains are
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seen in the terminal cell. In Linum usitatissimum such grains are equal in both basal and terminal cells (Bhat and Vijayaraghavan, 1980). The absence of polysaccharide grains during early embryogenesis is indicative of participation in the nutrition of the developing embryo. In Najas marina, polysaccharide grains appear in the cotyledonary and hypocotyledonary regions of the embryo proper, after the initiation of the embryonic shootapex and the embryonic leaves. This indicates that these regions act as repository of storage material. The zygote of Najas marina is a polarized cell with dense concentration of proteins and R N A at the chalazal end. A similar trend of distribution of these metabolites has been reported in m a n y Vanda cultivars (Alvarez and Sagawa, 1965) and Capsella bursa-pastoris (Schulz and Jensen, 1968a). In N. marina, the concentration of proteins and R N A at the 2-celled proembryo stage is more in the basal cell than in the terminal cell. The nuclei in both cells are rich in proteins and R N A . A higher concentration of proteins and nucleic acids is reported in the basal cell of Stellaria media (L.) Vill. (Pritchard, 1964), Capsella bursa.pastoris (Schulz and Jensen, 1968b) and Limnophyton obtusifolium Miq. (Shah and Pandey, 1978). In Najas marina, the single, vesicular, basal cell and the embryonal cells beginning from the globular proembryo stage are rich in proteins and nucleic acids. The same trend is seen in Stellaria media (Pritchard, 1964) and Capsella bursa-pastoris (Schulz and Jensen, 1968b). In N. marina, the presumptive sites of cotyledon and embryonic shoot-apex show more protein and RNA contents than other embryonal cells as compared with Capsella bursapastoris (Schulz and Jensen, 1968b), Petunia hybrida hort. (Vallade, 1970) and Limnophyton obtusifolium (Shah and Pandey, 1978).
The embryo suspensor The suspensor has been classically assigned the role of anchoring the embryo and pushing it to a favourable position in the embryo sac. The studies during recent years on early embryogenesis are made with special reference to development, structure and functions of the suspensor (Nagl, 1962, 1973, 1974). In Najas marina (present work), the suspensor cell wall subjacent to the nucellus stains deeply for PAS-reaction. Pritchard (1964) has also described the micropylar end of the primary suspensor cell in Stellaria media to be deeply covered with PAS-positive membrane and even suggested that carbohydrates m a y move through this area. Recent ultrastructural studies of the basal cell have revealed the presence of wall ingrowths in Phaseolus coccineus L. (Yeung and Clutter, 1978), P. vulgarisL. (Schnepf and Nagl, 1970), Diplotaxis erueoides (L.) DC. (Simoncioli, 1974), Tropaeolum majus L. (Nagl, 1976a) and Alyssum maritimum (L.) Lain. (Prabhakar and Vijayaraghavan, 1983). Nagl (1976b) postulated that the suspensor of taxa with storage cotyledons and underdeveloped endosperm grows by endoreduplication and polytinization and even grows faster than
56 the organogenetic part of the embryo. The present work on Najas marina supports this contention and further assigns an active role of absorption and translocation to the suspensor. High protein synthesis has been observed in the suspensor cell of Najas marina. The suspensor absorbs nutritive substances from the adjoining tissues and thus acts as a transfer cell (see also Gunning and Pate, 1969). The suspensor is the place of synthesis of growth regulating substances (Ponzi and Pizzolongo, 1973) and high phytohormone activity has been reported in the suspensor of Phaseolus coccineus (Cionini et al., 1976). It is interesting that the suspensor during early stages stores lipids whereas in later stages stores starch (Nagl, 1976b), According to Satina and Rietsema (1959), the suspensor cell of Datura sp. serves as a direct source of oil to the embryo. In Phaseolus coccineus, the disappearance of plastids from suspensor cells during early maturation has been considered to indicate that plastids play a specific role in embryo development (Yeung and Clutter, 1979). During final maturation of the embryo, the suspensor cell generally degenerates. The autolytic process of the suspensor cell of Phaseolus sp. and Tropaeolum sp. starts at the micropylar p o r t i o n (Nagl, 1976b). Early autophagy in Phaseolus coccineus is due to the appearance of cytolysomes (Villiers, 1967) which originate due to the fusion of endoplasmic reticulum segments. Multivesicular bodies are also associated with the autophagy in Tropaeolum majus (Nagl, 1976b). Autophagy is due to tonoplast invagination, intravacuolar bodies and myelin-like membranes. The orderly lysis of the suspensor cells in plant embryos negates the assumption that this organ is crushed due to embryo growth. Schulz and Jensen (1969) have even indicated the destruction of the protoplast in the suspensor cell of Capsella bursa-pastoris and thus converting the suspensor into a conducting tube. The present work on Najas marina and the findings of other investigators suggest that the suspensor undergoes a programmed lysis to provide food material to the embryo upto a stage when the embryo is able to store and provide its own food material for its development. In Najas, the single basal cell that acts also as suspensor cell lacks mitosis, undergoes only cell enlargement, is persistent, and undergoes partial autolysis. In addition to the other functions mentioned above, this cell acts as a barrier for the emergence of the primary root from the normal radicular end of the embryo. Thus, there is a shift in the root primordium to a lateral position as a result of which 3 or 4 epidermal cells that show polarized distribution of proteins and polysaccharide grains differentiate and act as the new root-primordium.
Nucleo--vytoplasmic interaction At the 3-leaf embryo stage, nucleolar bodies are seen in the basal cell cytoplasm of Najas marina. Such nucleolar bodies, also known as nucleolar
57 extrusions or nucleolus-like bodies (Nagl, 1970), contain proteins, RNA and are also feulgen-positive (see also Avanzi et al., 1970, 1972). The phenomenon of nucleo---cytoplasmic interaction is reported in several Lilium sp. where homogeneous material is extruded from nucleolus into cytoplasm (Flint and Johansen, 1958) and in the basal cell as well as in the chalazai endosperm cell in Potamogeton nodosus Poir. (Vijayaraghavan and Kapoor, 1985). The phenomenon of nucleo--cytoplasmic interaction as observed in the basal cell of Najas marina is due to differentiation and high metabolic activity. Storage bodies In Najas marina the food is stored as starch in the embryo proper. The hypocotyledonary and cotyledonary regions of the embryo are engorged with starch grains as observed in Phaseolus coccineus (Yeung and Clutter, 1978). In contrast, mature seeds of wheat (Hu, 1964), Linum usitatissimum and Ranunculus sceleratus (Dhar, 1976), Iberis amara and Alyssum maritimum (Prabhakar, 1979) show insoluble polysaccharide grains stored only as cell-wall materials. In Najas marina, the distribution of polysaccharide grains in different regions of the embryo is particularly interesting. Small polysaccharide grains appear at random in the root-primordium, procambium and embryonic shoot-apex. In hypocotyledonary and cotyledonary regions, the starch grains congregate around the nucleus. Thus there may be a physiological interaction between the plastids and the nucleus. Such grains in the epidermal cells show polarized distribution and in the initials of the root-primordium these are compactly clustered around the nucleus. Interestingly, the electron microscopic investigations on roots have emphasized the polarity of statocytes and participation of amyloplasts in geoperception (Haberkorn and Sievers, 1977). The hila of starch grains axe protein-positive and show pyroninophilia showing the presence of proteins and RNA (see also Badenhuizen, 1969). The distribution of starch in the various regions of the embryo is interesting and needs further investigation in other aquatic angiosperms. In traseminal germination In vivo intraseminal germination is a unique phenomenon in Najas marina. Under in vitro conditions attempts have been made to induce root/ shoot or develop embryos into plantlets. Plant embryos, in tissue culture, often exhibit precocious germination (Rijven, 1952). The high concentration of sucrose and mannitol is said to induce such germination in barley embryos (Ziebur et al., 1950). A distinction between intraseminal germination and embryogenesis should be made. In the former, the embryo acquires seedling characteristics as mitosis is restricted to meristems and primordia.
58 In normal embryogenesis: (i) cell division occurs throughout the embryo; (ii) cells are small in size; (iii)with little vacuolation and; (iv) intense cytoplasm, In Na]as marina, intraseminal germination is intrinsic in the seed and perhaps is an adaptive mechanism to ensure propagation of a future generation. Intraseminal germination is also of interest because it provides a basis to study and understand problems involved in seed dormancy. CONCLUSIONS
Besides anchoring the e m b r y o the embryo basal cell in Najas marina has other functions to play. It helps in the translocation of metabolites to the growing e m b r y o during early stages; and it acts at seed maturity as a barrier for the emergence o f the initial r o o t from the normal radicular end of the embryo. It is also envisaged that this p h e n o m e n o n initiates the development of embryonic shoot apex and embryonic leaves at the epicotylary end and thus promotes precocious germination. Thus, it appears that there is no seed d o r m a n c y in Najas marina and the intraseminal germination in this t a x o n is an adaptation for the hazardous life o f the aquatic habitat. The physiology and ultrastructural aspects involved in such a p h e n o m e n o n in aquatic plants are n o t well understood and need proper attention. ACKNOWLEDGEMENTS
We thank Professor R.N. Chopra for facilities. The material of Najas marina L. fixed by Dr. R. Raghuvanshi, Department o f Botany, University of Rajasthan, Jaipur, India, i s gratefully acknowledged. We thank Dr. S.J. Mayo and Dr. P.S. Green, Royal Botanic Gardens, Kew, Surrey, England, for identification of the material. We thank Professor Dr. C. den Hartog and an u n k n o w n reviewer for critically going through the manuscript and offering valuable suggestions. One of us (T.K.) is grateful to the Planning Unit, University of Delhi, for financial assistance. REFERENCES Alvarez, M.R. and Sagawa, Y., 1965. A histochemical study of embryo development in Vanda (Orchidaceae). Caryologia, 18: 251--261. Avanzi, S., Cionini, P.G. and D'Amato, F., 1970. Cytochemical and autoradiographic analyses on the embryo suspensor cells of Phaseolus coccineus. Caryologia, 23:605 ---638. Avanzi, S., Durante, M., Cionini, P.G. and D'Amato, F., 1972. Cytological localization of ribosomal cistrons in polytene chromosomes of Phaseolus coccineus. Chromosoma, 39: 191--203. Badenbuizen, N.P., 1969. The Biogenesis of Starch Granules in Higher Plants, New York, pp. 1--121. Bhat, U. and Vijayaraghavan, M.R., 1980. Distribution of insoluble polysaccharides in Linum usitatissimum -- Zygote to seedling. Cytologia, 45:65-75. Cionini, P.G., Bennici, A., Alpi, A. and D'Amato, F., 1976. Suspensor, gibberellin and in vitro development of Phaseolus coccineus embryos. Planta, 131 : 115--117.
59 Dhar, U., 1976. Part I. Histochemical Studies in Linum usitatissimum l.Ann, and Ranunculus sceleratus Linn. -- Egg to seedling. Part II. Embryology of Cyrilla racemiflora Linn. and Cliftonia monophylla (Lain.) Britton ex Sarg. with Special Reference to Systematic Position of Cyrillaceae. Ph.D. Thesis, Univ. Delhi, Delhi, pp. 1--101. Flint, F.F. and Johanscn, D.A., 1958. Nucleocytoplasmic relationships in the Fritillaria type of megagametogenasis. Am. J. Bot., 45: 464--473. Georgieva, I.D., 1965. Recherches au microscope ~ fluorescence des polysaccharides dans l'embryogen~se du Lilium. C.R. Acad. Bulg. Sci., 18: 477--480. Gunning, B.E.S. and Pate, J.S., 1969. "Transfer cells" -- Plant cells with wall ingrowths, specialized in relation to short distance transport of solutes -- their occurrence, structure and development. Frotoplasma, 68: 107--133. Haberkoru, H.R. and Sievers, A., 1977. Response to gravity of roots growing in water. Naturwissensch., 64: 639--640. Hu, S.Y., 1964. Accumulation and movement of starch during caryopsis development of wheat. Acta Bot. Sinica, 12: 139--148. Jensen, W.A., 1962. Botanical Histochemistry. Freeman, San Francisco. Jensen, W.A., 1968. Cotton embryogenesis: The zygote. Planta, 79: 346--366. Kallarackal, J., 1974. A modified Schiff reagent for use in Feulgen reaction. Curt. Sci., 43: 120--121. Maheshwari, P., 1950. An Introduction to the Embryology of Angiosperms. McGraw-Hill, New York, pp. 1--453. Mazia, D., Brewer, P.A. and Alfert, M., 1953. The cytochemical staining measurement of protein with mercuric bromophenol blue. Biol. Bull., 104: 57--67. Nagl, W., 1962. {~Poer Endopolyploidie, Restitutiormkernbildun~ und Kernstrukturen irn Suspensor yon Angiospermen und einer Gymnospermee. Ost. Bot. Z., 1 0 9 : 4 3 1 --494. Nagl, W., 1970. Temperature dependent functional structures in the polytene chromosomes of Phaseolus, with special reference to the nucleolus organizers. J. Cell Sci., 6: 87--107. Nagl, W., 1973. The angiosperm suspensor and the mammalian trophoblast: Organs with similar cell structure and function? Bull. Soc. Bot. Fr., 1973: 289--301. Nagl, W., 1974. The Phaseolus suspensor and its polytene chromosomes. Z. Pflanzenphysiol., 73: 1--44. Nagl, W., 1976a. Early embryogenesis in Tropaeolum majus L.: Ultrastructure of the embryo-suspensor. Biochem. Physiol. Pflanz., 170: 253--260. Nagl, W., 1976b. Ultrastructure and developmental aspects of autolysis in embryosuspensors. Bet. Dtsch. Bot. Ges., 89: 301--311. Ponzi, R. and Pizzolongo, P., 1973. Ultrastructure of plastids in the suspensor cells of Ipomoea purpurea Roth. J. Submicrosc. Cytol., 5 : 257--263. Prabhakar, K., 1979. Histochemical and Ultrastructural Studies in Some Cruciferae: Zygote to Seedling. Ph.D. Thesis, Univ. Delhi, Delhi, pp. 1 --151. Prabhakar, K. and Vijayaraghavan, M.R., 1983. Histochemistry and ultrastructure of suspensor cells in Alyssum maritimum. Cytologia, 48: 389--402. Pritchard, H.N., 1964. A cytochemical study of embryo development in Stellaria media. Am. J. Bot., 51: 472--479. Rijven, A.H.G.C., 1952. In vitro studies on the embryo of Capsella-bursa-pastoris. Acta Bot. Neerl., 1: 158--200. Satina, S. and Rietsema, J., 1959. Seed development. In: A.G. Avery, S. Satina and J. Rietsema (Editors), Blakeslee: The Genus Datum. New York, pp. 181--195. Schnepf, E. and Nagl, W., 1970. Uber elmge Strukturbesonder-heiten dex Suspensorzellen von Phaseolus vulgaris. Protoplasma, 69: 133--143. Schulz, S.P. and Jensen, W.A., 1969. Capsella embryogenesis: The suspensor and the basal cell. Protoplasma, 67: 139--163.
60 Schuiz, S.R. and Jensen, W.A., 1968a. CapseUa embryogencsis: The egg, zygote and young embryo. Am. J. Bot., 55: 807--819. Schulz, S.R. and Jensen, W.A., 1968b. Capsella embryogenesis: The early embryo. J. Ultrastmct. Res., 22: 376--392. Shah, C.K. and Pandey, S.N., 1978. Histochemical studies during embryogencsis in Limnophyton obtusifolium. Phytomorphology, 28: 31--42. Simoncioli, C., 1974. Ultrastructure characteristics of Diplotaxis erucoides (L.) DC.: Suspensor. G. Bot. Ital., 108: 175--189. Tepper, H.B. and Gifford, E_M., Jr., 1962. Detection of ribonucleic acid with pyronin. Stain Technol., 37: 120--121. Vallade, J., 1970. D~veloppment embryonnaire chez un Petunia hybrida Hort. C.R. Hebd. S~anc. Acad. Sci., Paris, 270: 1893--1896. Vijayaraghavan, M.R. and Bhat, U., 1982. Localization of macromolecules during achene development in Ranunculus sceleratus L. Beitr. Biol. Pflanzen, 57 : 105--118. Vijayaraghavan, M.R. and Kapoor, T., 1985. Nucleo-cytoplasmic interaction in the embryo-basal cell and in the chalazal endospermchamber cell in Potamogeton nodosus. Cytologia, in press. Villiers, T.A., 1967. Cytolysomes in long-dormant plant embryo cells. Nature (London), 214: 1356--1357. Yeung, E.C. and Clutter, M.E., 1978. Embryogeny of Phaseolus coecineus: Growth and microanatomy. Protoplasma, 94: 19--40. Yeung, E.C. and Clutter, M.E., 1979. Embryogeny of Phaseolus coccineus: The ultrastructure and development of the suspensor. Can. J. Bot., 57: 120--136. Ziebur, N.K., Brink, R.A., Graf, L.H. and Stahmar, M.H., 1950. The effect of casein hydrolysate on the growth in vitro of immature Hordeum embryos. Am. J. Bot., 37: 144--148.
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Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
EMBRYOGENESIS HISTOCHEMICAL
IN NAJA8 MARINA L.: STRUCTURAL APPROACH
AND
M.R. V I J A Y A R A G H A V A N and TRIPAT KAPOOR
Department of Botany, University of Delhi, Delhi-110007 (India) (Accepted 15 March 1985)
ABSTRACT
Vijayaraghavan, M.R. and Kapoor, T., 1985. Embryogenesis in Najas marina L.: Structural and hlstochemicalapproach. Aquat. Bot., 22: 45--60. In Najas marina L. few polysaccharide grains are observed in zygote, basal cell and embryonal cells until the initiation of embryonic shoot-apex. With the formation o f the shoot-apex, numerous polysaccharide grains engorge in the embryonal cells. The basal cell wall, subjacent to the nucellus, stains intensely with PAS (Periodic Schiff's)-reaction. The concentration of proteins and R N A increases in the basal cell. Interestingly, the e m b r y o shows intraseminal germination. The cells o f embryonic shoot-apex, embryonic leaves, r o o t primordium and procambial cells show a few polysaccharide grains while the cells of h y p o c o t y l e d o n a r y and cotyledonary regions are engorged with polysaccharide (starch) grains. Uniform distribution o f proteins and RNA is observed in the embryonic shoot-apex, embryonic-leaves, root primordium and procambium, but the cells of h y p o c o t y l e d o n a r y and c o t y l e d o n a r y zones exhibit a low profile for these metabolites. The initial root-primordium remains quiescent. Three or 4 epidermal cells, subjacent to this quiescent primordium, differentiate; show densely stained, polarised, protein bands; and act as the future r o o t primordium. The nucleus of the basal cell becomes p o l y p l o i d and densely stains for proteins, RNA and DNA. At the globular p r o e m b r y o stage, numerous nucleolar bodies migrate towards the periphery of the nucleus and at the 3-leaf e m b r y o stage, these nucleolar bodies, rich in proteins and RNA, are located in the cytoplasm revealing nucleo-cytoplasmic interaction. The basal cell that never divides, but only enlarges, is persistent in the mature seed.
INTRODUCTION The information on structural development of aquatic monocotyledonous embryogenesis is meagre (Maheshwari, 1950). Little work exists on the cytochemical aspects of aquatic angiosperm embryogenesis. Therefore, the present work on Najas marina L. was undertaken to study the distribution pattern of various metabolites during embryogenesis and intraseminal germination.
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© 1985 Elsevier Science Publishers B.V.
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MATERIAL AND METHODS Post-fertilized flowers and fruits (at various stages of development) of Naias Marina were fixed in FAA (Formalin Acetic Acid) and AA (Acetic Alcohol). The fixed material was dehydrated in a tertiary-butyl-alcohol series and embedded in paraffin wax. Twelve gm sections were cut and mounted on chemically-cleaned slides using Haupt's adhesive. The material that was fixed in FAA was used for the localization of insoluble polysaccharides and proteins, and that fixed in AA was used for nucleic acids. Insoluble polysaccharides were localized with the PAS (Periodic Schiff's) technique (Jensen, 1962), proteins with the mercuric-bromophenol blue method (Mazia et al., 1953), RNA by the Pyronin-Y method (Tepper and Gifford, 1962) and DNA by the Feulgen reaction (Kallarackal, 1974). Control slides were made in order to check the specificity of the various reactions. Starch grains were confirmed by IKI (Potassium Iodide) test. RESULTS
Structural aspects Najas marina (Fig. 1A) is a dioecious plant. The female flower is devoid of a perianth. The gynoecium is unilocular and uniovulate. The style and stigma are conspicuous in the fruit (Fig. 1B). The zygote (Fig. 2A) divides transversely resulting in a large basal cell and a small terminal cell. The basal cell remains undivided and functions directly as the embryo suspensor cell whereas the terminal cell undergoes numerous mitotic divisions and forms the organogenetic part of the embryo. Further growth and differentiation in this part of the embryo result in globular (Fig. 3A) and mature embryos. The embryo development follows the conventional Caryophyllad type (Maheshwari, 1950). Intraseminal germination The embryo does not undergo a dormant phase/after-ripening period and germinates precociously to produce a well developed embryonic shootapex (Figs. 2F, 4C) and upto 3 leaves (Fig. 2E) within the seed. The embryo thus reveals the following features: (1) basal cell; (2) root primordium; Fig. 1A--H. Na]as marina, external morphology (A and B), localization of insoluble polysaccharides (C--H); (A) Vegetative portion of female plant X 6; (B) A fruit showing persistent stigma (s) x 12; (C,D,G) Portions of embryo at 3 leaf stage showing root primordium (r) with basal cell (bc), embryonic shoot-apex (pv) and cotyledonary tip (c), respectively: C × 200; D × 450; G X 390; (E) Three epidermal cells (arrows) that differentiate are the new root initials. The polysaccharide grains cluster around nuclei (nu) in these cells x 390; (F,H) A few cells from hypocotyledonary and cotyledonary regions. The epidermal cells (ed) in H show polarized distribution of polysaccharide grains F X450, H X 390.
47
A
,~. 2A--F. Najas marina, localization of total proteins (A,C--E) and RNA (B,F); (A) gote (z), persistent synergid (sy) and nucellar cells (n) are protein rich × 270; (B) .~globular embryo showing basal cell (bc) and organogenetic portion rich in RNA 270; (C,D) Preglobular and globular embryos stained densely for proteins. Inset in shows the basal cell at globular embryo stage × 200; (E) Portion of embryo showing bryonic shoot-apex (pv) and 3 embryonic leaves (1) × 135; (F) Portion of seed showing raseminal germination. The cells of root primordium (r), procambium (pl), embryonic )ot-apex and embryonic leaf stain uniformly for RNA and cells of hypocotyledonary ;), cotyledonary (c) zones and persistent nucellus (pr) are poor in cytoplasmic RNA. e degenerating chalazal endosperm chamber (cc) persists × 100.
49
°4P
9
" , ~ I bc
B
A
Q
D.
E ~IIB -
.'t
m
•
Fig. 3A--H. Najas marina, localization of DNA; A--D. Basal cell (bc) and embryonal cells during progressive advancing stages of embryogeny × 200; (E) Portion of mature embryo, and a few cells of procambium (pl) x 390; (F) Cells of embryonic shoot-apex (pv) and embryonic leaf (1) with DNA-rieh nuclei x 390; (G) Portion of cotyledon to show that hila (arrows) are feulgen-positive. Binary fission of the hilum is noteworthy x 850; (H) Portion of e m b r y o at 3-leaf stage to show that the intensity of DNA staining is reduced in the nuclei of cotyledonary cells (c) x 200.
50
I
51
(3) hypocotyledonary zone; (4) cotyledonary region; (5) procambium; (6) embryonic shoot-apex; (7) embryonic leaves. The cells of root primordium (Fig. 2F), procambium (Fig. 2F), embryonic shoot-apex (Fig. 2F) and embryonic leaves (Fig. 2E) are small and have uniform distribution of cytoplasm whereas the cells of hypocotyledonary and cotyledonary (Fig. 2F) regions are large and vacuolate with thin cytoplasmic contents. Embryo basal cell
The basal cell develops into a vesicular structure (Fig. 2D inset). The nucleus becomes polyploid. At about the 3-1ear embryo stage, prominent nucleolar-blebs (Fig. 4G, arrows) surrounded by hyaline areas are seen in the cytoplasm. The basal cell is persistent (Figs. 1C, 4B) in the mature seed and perhaps prevents the emergence of the original root-primordium from the radicular end. Therefore, the new root-primordium (Fig. 4D) differentiates from the epidermal cells located toward the lateral sides of the original root-primordium. Testa and pericarp
At zygote and preglobular embryo stages, the inner integument is 2layered except at the micropylar end where it is 3-layered (Fig. 5A). During later progressive advancing stages of embryo development, these 2 layers, except at the micropylar end, become compressed. The outer integument, at the zygote stage, comprises 4 or 5 layers of cells. The cells of the outer epidermis expand radially and tangentially, whereas the other layers increase in number (Fig. 5B). At the 3-embryonic leaf stage, 7 layers of cells sandwiched between the 2 epidermes develop sclerenchymatous cell wall thickenings and pittings (Figs. 5C--E). The pericarp (Fig. 5B) except at the chalazal end, remains thin and 2-layered and shows little morphological or histological changes throughout the embryogenesis.
Fig. 4A--H. Najas marina, localization o f total proteins ( A - - F ) and R N A (G,H); (A,B) Portions of embryos to show cells o f r o o t primordium (r) that stain uniformly for cytoplasmic and nuclear proteins. The basal cell (bc) is rich both in cytoplasmic and nuclear proteins A X 100, B × 400; (C) Cells o f embryonic shoot-apex (pv) and embryonic leaves (l) showing homogeneous distribution of proteins X 400; (D) A few epidermal cells of the h y p o c o t y l e d o n a r y region that show polarised protein bands (arrows), are the new root primordium initials × 400; (E) Portion of the cotyledonary cells (c) × 400; (F) A few h y p o c o t y l e d o n a r y cells showing procambium (pl) x 400; (G) Basal cell at 3-leaf embryo stage to show pyroninophilic nucleolar-blebs (arrows) and nucleolus X 900; (H) A few cotyledonary cells showing congregation of starch grains around the nuclei (nu), hila (arrows) are pyroninophilic x 900.
52
3 Fig. 5A--E. Najas marina, localization of insoluble polysaccharides; (A) Testa at preglobular embryo stage. Inner integument (ii) is 3-layered at micropylar region × 200; (B) Longisection of fruit at embryo (em) stage. The pericarp (pc) is 2-layered x 80; (C) Portion of longisection of fruit at 3-leaf embryo stage x 200; (D,E) A few cells from the outer integument (oi) at 3-leaf embryo stage showing secondary cell wall thickenings (arrows), except epidermes (ed), pittings and polysaccharide grains (ps). In E, pittings are seen in surface view x 400.
Histochemical aspects Insoluble polysaccharides The z y g o t e , preglobular- and g l o b u l a r - e m b r y o s s h o w small p o l y s a c c h a r i d e grains. W h e n t h e e m b r y o n i c s h o o t - a p e x is well d e v e l o p e d , small p o l y s a c charide grains are localized in h y p o c o t y l e d o n a r y a n d c o t y l e d o n a r y cells,
53
but the cells of the root primordium, procambium and embryonic shootapex are devoid of such grains. The root primordium, procambium and embryonic shoot~pex, at the l-leaf embryo stage, have few polysaccharide grains, whereas the cells of hypocotyledonary and the cotyledonary regions are engorged with this metabolite. However, at the 3-leaf embryo stage root primordium (Fig. 1C), embryonic shoot-apex (Fig. 1D), procambium and embryonicleaves reveal small polysaccharide grains while the cells of hypocotyledonary (Fig. 1F) and cotyledonary (Fig. 1G) regions have copious deposition of starch grains. The alignment of such polysaccharide grains in various regions of the embryo is interesting. In root primordium (Fig..1C), procambium and embryonic shoot-apex (Fig. 1D) small polysaccharide grains are seen at random. In hypocotyledonary (Fig. 1F) and cotyledonary (Fig. 1G) regions, the polysaccharide grains (starch) congregate around the nucleus. In the epidermal cells such grains show polarized distribution (Fig. 1H) and are restricted to the abaxial side. Three or 4 epidermal cells, adjacent to the hypocotyledonary r e g i o n a n d below the root primordium, differentiate as the new future root primordium initials, and contain polysaccharide grains compactly clustered around the nucleus in each cell (Fig. 1E). The embryo basal cell (Fig. 1C) forms a cap-like structure over the radicular end, and throughout embryogenesis shows an intense PAS-positive cell wall, but lacks polysaccharide grains. During progressive advancing stages of embryogenesis, few polysaccharide grains are localized in the nucellus. At the 3-leaf embryo stage, the nucellar cells that persist at the micropylar, chalazal and the lateral sides flanking the embryo constitute the perisperm and are faintly stained. The cells of the inner integument (Fig. 5A), at preglobular and globular embryo stages, are devoid of polysaccharide grains. The cells of the outer integument, except the outer and the inner epidermes, reveal polysaccharide grains and at about the mature embryo stage, the polysaccharide contents increase enormously (Fig. 5B). At the 3-leaf embryo stage, prominent, PAS-positive, cell-wall thickenings (Figs. 5C--E) and abundant polysaccharide grains (Figs. 5D,E) are present in all layers of the outer integument except the epidermes (Fig. 5C). The cell walls of the pericarp are PASpositive (Fig. 5B), but the cells lack polysaccharide grains during embryogenesis. Proteins and nucleic acids
The zygote (Fig. 2A) is rich in proteins and RNA. Further, at octant, globular (Figs. 2B--D) and mature embryo stages, the basal cell and the embryonal cells are protein and RNA rich. The zygote nucleus stains feebly for DNA. At the 2-ceUed pro-embryo stage, the nucleus of the terminal cell stains with lesser intensity than that of the basal cell. The nuclei of embryonal cells at globular (Fig. 3A) and mature (Fig. 3B) embryo stages are rich in DNA content. The basal cell nucleus, during embryogenesis,
;ains densely for DNA (Figs. 3A--D) when compared to the embryonal ells. At 1--3-leaf embryo stages, cells of root-primordium (Fig. 4A), embryonic ~oot-apex (Fig. 4C), embryonic leaves (Fig. 2E) and procambium (Fig. F) stain uniformly for proteins (Figs. 4A,C,F) and RNA (Fig. 2F). The ells of the hypocotyledonary (Fig. 4F) and the cotyledonary (Fig. 4E) reions have dense nuclear proteins (Figs. 4E,F) and RNA (Fig. 2F), but the ytoplasmic proteins and RNA are less. The proteins in these cells are fibrillar i nature (Fig. 4F). Three or four epidermal cells (arrows) subjacent the riginal root-primordium differentiate, show densely-stained and polarized rotein bands (Fig. 4D) and act as the new initials of the future root priLordium. At the l-leaf embryo stage, nuclei of root primordium (Fig. C), procambium (Fig. 3E), embryonic shoot-apex and leaf (Fig. 3F), ; well as the embryonal cells stain for DNA. The nuclei of hypocoty~donary and cotyledonary (Fig. 3H) cells, at 3-leaf embryo stage, show ~ss intensity for DNA. The starch grains present in these cells have Feulgenositive (Fig. 3G) and pyroninophilic hila (Fig. 4H). Hila of such grains ~hibit binary fission (Fig. 3G). The basal cell at this stage is partially Jtolytic, but persistent and reveals 3--6 nucleolar blebs which axe pro~,in-rich and pyroninophilic (Fig. 4G). The nucellar cells at zygote (Fig. 5A), preglobular (Fig. 2C) and globular nbryo stages, are protein and RNA rich, whereas at the mature embryo age, the intensity of the protein and the RNA contents is very low. owever, the cells of the 3-layered nucellus that persist at the micropylar ~gion stain for nuclear proteins and nucleolar RNA, but those cells that ank the embryo are poor both in protein and RNA (Fig. 2F) contents ld even their nuclei stain feebly for DNA. The cells of integuments and ~ricarp reveal a decreasing trend of protein and RNA contents during nbryogenesis. [SCUSSION ygote and embryo In Najas marina the polysaccharide grains are absent around the zygote Jcleus. This is in contrast to the accumulation of polysaccharide grains ,en around the zygote nucleus in Gossypium hirsutum L. (Jensen, 1968), zpsella bursa-pastoris (L.) Medic. (Schulz and Jensen, 1968a), Iberis amara . (Prabhakar, 1979) and Linum usitatissimum L. (Bhat and Vijayaraghavan, ~80). At the 2~elled, preglobular and globular proembryo stages no polyccharide grains are localized in the basal cell and embryonal cells of Najas arina. In Capsella bursa-pastoris (Schulz and Jensen, 1968a), at the 2~lled stage, polysaccharide grains are more in the basal cell than in the rminal cell, but in Lilium spp. (Georgieva, 1965) and Ranunculus sceletus L. (Vijayaraghavan and Bhat, 1982) more polysaccharide grains are
55
seen in the terminal cell. In Linum usitatissimum such grains are equal in both basal and terminal cells (Bhat and Vijayaraghavan, 1980). The absence of polysaccharide grains during early embryogenesis is indicative of participation in the nutrition of the developing embryo. In Najas marina, polysaccharide grains appear in the cotyledonary and hypocotyledonary regions of the embryo proper, after the initiation of the embryonic shootapex and the embryonic leaves. This indicates that these regions act as repository of storage material. The zygote of Najas marina is a polarized cell with dense concentration of proteins and R N A at the chalazal end. A similar trend of distribution of these metabolites has been reported in m a n y Vanda cultivars (Alvarez and Sagawa, 1965) and Capsella bursa-pastoris (Schulz and Jensen, 1968a). In N. marina, the concentration of proteins and R N A at the 2-celled proembryo stage is more in the basal cell than in the terminal cell. The nuclei in both cells are rich in proteins and R N A . A higher concentration of proteins and nucleic acids is reported in the basal cell of Stellaria media (L.) Vill. (Pritchard, 1964), Capsella bursa.pastoris (Schulz and Jensen, 1968b) and Limnophyton obtusifolium Miq. (Shah and Pandey, 1978). In Najas marina, the single, vesicular, basal cell and the embryonal cells beginning from the globular proembryo stage are rich in proteins and nucleic acids. The same trend is seen in Stellaria media (Pritchard, 1964) and Capsella bursa-pastoris (Schulz and Jensen, 1968b). In N. marina, the presumptive sites of cotyledon and embryonic shoot-apex show more protein and RNA contents than other embryonal cells as compared with Capsella bursapastoris (Schulz and Jensen, 1968b), Petunia hybrida hort. (Vallade, 1970) and Limnophyton obtusifolium (Shah and Pandey, 1978).
The embryo suspensor The suspensor has been classically assigned the role of anchoring the embryo and pushing it to a favourable position in the embryo sac. The studies during recent years on early embryogenesis are made with special reference to development, structure and functions of the suspensor (Nagl, 1962, 1973, 1974). In Najas marina (present work), the suspensor cell wall subjacent to the nucellus stains deeply for PAS-reaction. Pritchard (1964) has also described the micropylar end of the primary suspensor cell in Stellaria media to be deeply covered with PAS-positive membrane and even suggested that carbohydrates m a y move through this area. Recent ultrastructural studies of the basal cell have revealed the presence of wall ingrowths in Phaseolus coccineus L. (Yeung and Clutter, 1978), P. vulgarisL. (Schnepf and Nagl, 1970), Diplotaxis erueoides (L.) DC. (Simoncioli, 1974), Tropaeolum majus L. (Nagl, 1976a) and Alyssum maritimum (L.) Lain. (Prabhakar and Vijayaraghavan, 1983). Nagl (1976b) postulated that the suspensor of taxa with storage cotyledons and underdeveloped endosperm grows by endoreduplication and polytinization and even grows faster than
56 the organogenetic part of the embryo. The present work on Najas marina supports this contention and further assigns an active role of absorption and translocation to the suspensor. High protein synthesis has been observed in the suspensor cell of Najas marina. The suspensor absorbs nutritive substances from the adjoining tissues and thus acts as a transfer cell (see also Gunning and Pate, 1969). The suspensor is the place of synthesis of growth regulating substances (Ponzi and Pizzolongo, 1973) and high phytohormone activity has been reported in the suspensor of Phaseolus coccineus (Cionini et al., 1976). It is interesting that the suspensor during early stages stores lipids whereas in later stages stores starch (Nagl, 1976b), According to Satina and Rietsema (1959), the suspensor cell of Datura sp. serves as a direct source of oil to the embryo. In Phaseolus coccineus, the disappearance of plastids from suspensor cells during early maturation has been considered to indicate that plastids play a specific role in embryo development (Yeung and Clutter, 1979). During final maturation of the embryo, the suspensor cell generally degenerates. The autolytic process of the suspensor cell of Phaseolus sp. and Tropaeolum sp. starts at the micropylar p o r t i o n (Nagl, 1976b). Early autophagy in Phaseolus coccineus is due to the appearance of cytolysomes (Villiers, 1967) which originate due to the fusion of endoplasmic reticulum segments. Multivesicular bodies are also associated with the autophagy in Tropaeolum majus (Nagl, 1976b). Autophagy is due to tonoplast invagination, intravacuolar bodies and myelin-like membranes. The orderly lysis of the suspensor cells in plant embryos negates the assumption that this organ is crushed due to embryo growth. Schulz and Jensen (1969) have even indicated the destruction of the protoplast in the suspensor cell of Capsella bursa-pastoris and thus converting the suspensor into a conducting tube. The present work on Najas marina and the findings of other investigators suggest that the suspensor undergoes a programmed lysis to provide food material to the embryo upto a stage when the embryo is able to store and provide its own food material for its development. In Najas, the single basal cell that acts also as suspensor cell lacks mitosis, undergoes only cell enlargement, is persistent, and undergoes partial autolysis. In addition to the other functions mentioned above, this cell acts as a barrier for the emergence of the primary root from the normal radicular end of the embryo. Thus, there is a shift in the root primordium to a lateral position as a result of which 3 or 4 epidermal cells that show polarized distribution of proteins and polysaccharide grains differentiate and act as the new root-primordium.
Nucleo--vytoplasmic interaction At the 3-leaf embryo stage, nucleolar bodies are seen in the basal cell cytoplasm of Najas marina. Such nucleolar bodies, also known as nucleolar
57 extrusions or nucleolus-like bodies (Nagl, 1970), contain proteins, RNA and are also feulgen-positive (see also Avanzi et al., 1970, 1972). The phenomenon of nucleo---cytoplasmic interaction is reported in several Lilium sp. where homogeneous material is extruded from nucleolus into cytoplasm (Flint and Johansen, 1958) and in the basal cell as well as in the chalazai endosperm cell in Potamogeton nodosus Poir. (Vijayaraghavan and Kapoor, 1985). The phenomenon of nucleo--cytoplasmic interaction as observed in the basal cell of Najas marina is due to differentiation and high metabolic activity. Storage bodies In Najas marina the food is stored as starch in the embryo proper. The hypocotyledonary and cotyledonary regions of the embryo are engorged with starch grains as observed in Phaseolus coccineus (Yeung and Clutter, 1978). In contrast, mature seeds of wheat (Hu, 1964), Linum usitatissimum and Ranunculus sceleratus (Dhar, 1976), Iberis amara and Alyssum maritimum (Prabhakar, 1979) show insoluble polysaccharide grains stored only as cell-wall materials. In Najas marina, the distribution of polysaccharide grains in different regions of the embryo is particularly interesting. Small polysaccharide grains appear at random in the root-primordium, procambium and embryonic shoot-apex. In hypocotyledonary and cotyledonary regions, the starch grains congregate around the nucleus. Thus there may be a physiological interaction between the plastids and the nucleus. Such grains in the epidermal cells show polarized distribution and in the initials of the root-primordium these are compactly clustered around the nucleus. Interestingly, the electron microscopic investigations on roots have emphasized the polarity of statocytes and participation of amyloplasts in geoperception (Haberkorn and Sievers, 1977). The hila of starch grains axe protein-positive and show pyroninophilia showing the presence of proteins and RNA (see also Badenhuizen, 1969). The distribution of starch in the various regions of the embryo is interesting and needs further investigation in other aquatic angiosperms. In traseminal germination In vivo intraseminal germination is a unique phenomenon in Najas marina. Under in vitro conditions attempts have been made to induce root/ shoot or develop embryos into plantlets. Plant embryos, in tissue culture, often exhibit precocious germination (Rijven, 1952). The high concentration of sucrose and mannitol is said to induce such germination in barley embryos (Ziebur et al., 1950). A distinction between intraseminal germination and embryogenesis should be made. In the former, the embryo acquires seedling characteristics as mitosis is restricted to meristems and primordia.
58 In normal embryogenesis: (i) cell division occurs throughout the embryo; (ii) cells are small in size; (iii)with little vacuolation and; (iv) intense cytoplasm, In Na]as marina, intraseminal germination is intrinsic in the seed and perhaps is an adaptive mechanism to ensure propagation of a future generation. Intraseminal germination is also of interest because it provides a basis to study and understand problems involved in seed dormancy. CONCLUSIONS
Besides anchoring the e m b r y o the embryo basal cell in Najas marina has other functions to play. It helps in the translocation of metabolites to the growing e m b r y o during early stages; and it acts at seed maturity as a barrier for the emergence o f the initial r o o t from the normal radicular end of the embryo. It is also envisaged that this p h e n o m e n o n initiates the development of embryonic shoot apex and embryonic leaves at the epicotylary end and thus promotes precocious germination. Thus, it appears that there is no seed d o r m a n c y in Najas marina and the intraseminal germination in this t a x o n is an adaptation for the hazardous life o f the aquatic habitat. The physiology and ultrastructural aspects involved in such a p h e n o m e n o n in aquatic plants are n o t well understood and need proper attention. ACKNOWLEDGEMENTS
We thank Professor R.N. Chopra for facilities. The material of Najas marina L. fixed by Dr. R. Raghuvanshi, Department o f Botany, University of Rajasthan, Jaipur, India, i s gratefully acknowledged. We thank Dr. S.J. Mayo and Dr. P.S. Green, Royal Botanic Gardens, Kew, Surrey, England, for identification of the material. We thank Professor Dr. C. den Hartog and an u n k n o w n reviewer for critically going through the manuscript and offering valuable suggestions. One of us (T.K.) is grateful to the Planning Unit, University of Delhi, for financial assistance. REFERENCES Alvarez, M.R. and Sagawa, Y., 1965. A histochemical study of embryo development in Vanda (Orchidaceae). Caryologia, 18: 251--261. Avanzi, S., Cionini, P.G. and D'Amato, F., 1970. Cytochemical and autoradiographic analyses on the embryo suspensor cells of Phaseolus coccineus. Caryologia, 23:605 ---638. Avanzi, S., Durante, M., Cionini, P.G. and D'Amato, F., 1972. Cytological localization of ribosomal cistrons in polytene chromosomes of Phaseolus coccineus. Chromosoma, 39: 191--203. Badenbuizen, N.P., 1969. The Biogenesis of Starch Granules in Higher Plants, New York, pp. 1--121. Bhat, U. and Vijayaraghavan, M.R., 1980. Distribution of insoluble polysaccharides in Linum usitatissimum -- Zygote to seedling. Cytologia, 45:65-75. Cionini, P.G., Bennici, A., Alpi, A. and D'Amato, F., 1976. Suspensor, gibberellin and in vitro development of Phaseolus coccineus embryos. Planta, 131 : 115--117.
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