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The scutellar epithelium of rice seeds shows a variety of activities under different physiological conditions. The epithelial cells cease activity soon after the start of germination, but in the seminal root culture system, they retain their haustorial property for a long period. Furthermore, on solid medium with added 2, 4-D, they divide to form callus masses that lack a cuticular layer. The two-step method of callus culture results in a higher frequency of shoot-bud formation that is related to topographical variation on the callus surface. The development of a compact callus with white and green patches is a morphological prerequisite for shoot-bud formation. The cells constituting a callus mass behave differently according to the positional information determined by the internal and external environment. The external appearance of the callus largely depends on the extracellular matrix. The callus surface consists of a fragile slime-like membrane, firm fibrils, and a thin epicuticular layer. Finally, it is suggested that the newly formed cuticular layer on the callus surface is essential for shoot-bud formation. Consequently, we propose that the topographical characteristics of the callus mass play an important role in shoot-bud formation.

Before anthesis, rice antipodal cells are located between nucellar cells and a central cell, and have a large-sized lobed nucleus and many cell-wall ingrowths abutting the nucellar cells. An outstanding feature in the cytoplasm is the appearance of a multilayered, concentric configuration in endoplasmic reticula. They are constituted of several rings which enclose a component of cytoplasm or a plastid in the center. Two or three rings are connected with each other through a small joint, and as a result, a continuous lumen is ensured. These complicated figures of endoplasmic reticula probably come apart just after anthesis and rearrange into a great number of singular cisternae, of which features have been illustrated. From these circumstances, it is concluded that the configuration of endoplasmic reticula in antipodal cells strictly depends upon the developmental stages of the embryo sac. Furthermore, the dilation of lumens is observed on the singular cisternae located near the cell wall separating the central cell. The deposition and dilation of the singular segments are meaningful in relation to the nutrient transfer from the antipodal cells into the central cell. Finally, the higher activity of rice antipodal cells is indicated from the ultrastructural viewpoint. Additionally, the configurational change of endoplasmic reticula is discussed in the context of metabolic activities in the antipodal cells of rice and other plants.

Two groups of secretory glands, epidermal and mesophyll types, were examined in the young leaves of patchouli plants with light and electron microscopes. The structural characteristics of the component cells and features of the neighboring cells were clarified at the cyto-histological aspect. Glandular trichomes originate from both the adaxial and abaxial epidermis. Considerable attention is focused on internal, mesophyll glands arising in the palisade tissue. The secretory cell of the mesophyll glands is covered by a cuticular layer, and secretory materials are collected in a subcuticular space made between the cuticle and wall materials. In the vicinity of the glands, a vascular system occurs in spongy parenchyma. It includes vessel elements, sieve elements, large-sized cells of vascular bundle Sheath and morphologically specialized idioblasts; among them, relatively small sized-cells are present. The idioblasts have highly branched plasmodesmata in the wall and numerous mitochondria in the cytoplasm, but are lacking in a large, central vacuole. The branched plasmodesmata occur in groups. They intrude into the walls of sieve elements running in the interior of the vascular bundles and also into the walls of enlarged cells forming the bundle sheath. From a spatial viewpoint, it is likely that the vascular system has a relation to the transfer of nutrients into the mesophyll glands from the surrounding cells with photosynthetic activities. On the basis of these observations, the peculiar morphology of the mesophyll glands is discussed in terrns of their location and function in patchouli leaf blades.

In a rice embryo sac, there is an antipodal mass located at the opposite side of the micropyle, directly adjoining nucellar cells. Immediately after anthesis, the antipodal cells are characterized by the presence of numerous rough endoplasmic reticula and the prominent ingrowths of the wall parietal to nucellar cells. Additionally, they contain large-sized and irregularly lobed nuclei. The lobed parts of the nucleus are connected by narrow and short bridges composed of nucleoplasm and surround part of the cytoplasm, in which many plastids, mitochondria and rough endoplasmic reticula are evident. Most cell organelles are evenly distributed in cytoplasm around the nucleus and the terminal ends of endoplasmic reticula occasionally join the wall ingrowths protruding from the nucellus side. Some round-shaped structures with attached ribosomes are present in antipodal cytoplasm near the wall bordering a central cell. The nucellar cells in the neighborhood of the antipodal mass are already beginning to degenerate. It is proposed that this behaviour of antipodal cells and cell organelles is implicated in the possible role of the antipodals in apoplastic movement of nutrients from nucellar cells into a central cell in the embryo sac. Furthermore, we briefly describe the various routes of nutrient transfer in rice embryo sac.

The development of the radicle at the basal portion of rice rygotic embryos was studied. Median sagittal sections and transverse sections indicated that a radicle tip arose at the ventral side of the embryo 5 DAA (days after anthesis), apparently inducing the dorsiventrality of the embryo. A radicle was connected to a shoot tip by curved vascular strands, and the distance between the radicle tip and the shoot tip was about 300μm when measured in a straight line. It was demonstrated that the radicle contained root cap, root epidermis, cortex and central cylinder. The radicle's subapical initials originated a root cap, which was formed of more-or-less regular columellae of cells. Coleorhiza enveloped columnar root cap, by which the radicle tip was shown to comes first endogenously. The lateral surface of radicles was covered by a mucilaginous thick cell wall. Plasmodesmata were frequently seen in the cell walls of the cap and the epidermis, and additionally, in the walls separating the cap from thc epidermis. The position of embryos in the ovarian cavity in relation to the nucellar epidermis was transferred from the central to ventral side before these events. The initiation of shoot and root poles and the embryo size at the time are discussed in the context of the similarity of somatic embryoids to zygotic embryos.

Preparation methods for scanning electron microscopy (SEM) were studied with petunia and rice protoplasts. Fixation schedules to sustain protoplast size during alcohol dehydration were examined. When the different fixations were compared, the decrease in protoplast volume was observed to a variable extent during the dehydration process. The extent of volume decrease was reduced in order of glutaraldehyde, glutaraldehyde-osmium tetroxide, and glutaraldehyde-tannic acid-osmium tetroxide schedules. When fixed only with glutaraldehyde, the green color of the chloroplast in petunia leaf protoplasts was lost during alcohol dehydration. Well-defined scanning electron micrographs of petunia and rice protoplasts were obtained using a glutaraldehyde-tannic acid-osmium tetroxide schedule.

summary Embryogenically competent callus was induced from in vitro grown Saccharum officinarum L. (NCo 310) plantlets by plating young leaf segments on MS basal medium supplemented with 30 μM dicamba for 21 d in darkness, and subsequently transferring to a medium (EFDM) on which embryos could form and develop. Embryogenic response was improved if the EFDM was supplemented with 1 or 5 μM dicamba, casein hydrolysate (1 g 1−1), and 6% maltose or 6–9% corn syrup. Calli maintained their embryogenic competence for up to 16 months under these conditions, and with alternate weekly subcultures on to fresh media containing 20 μM and 30 μM dicamba in the dark, embryogenic callus proliferation could be further increased. Additional improvements in embryogenic competence were optimized to over 40 months when primary callus was alternately precultured at weekly intervals either with 10-5 M ABA or 5% sorbitol and then transferred to a medium containing 20 or 30 μM dicamba during maintenance. In general, medium plant growth regulator and nutritional cues, especially dicamha concentration, maltose/corn syrup levels and ABA/sorbitol preconditioning, were observed to influence critically both embryogenic callus proliferation and the frequency of somatic embryogenesis. The data are discussed within the context that these treatments can be successfully manipulated to enhance long-term embryogenic competence and high-frequency somatic embryogenesis in sugarcane.

Transmission electron microscopy revealed that a shoot apical meristem showed the first sign of organization at 3.5 DAA (days after anthesis), soon after an indentation by an incipient coleoptile on the upper ventral surface of the rice embryo. The demarcation of the meristem was terminated by a small depression on the lower surface at 4 DAA and then by periclinal divisions at 4.5 DAA. After that, the tip of the coleoptile gradually covered the apical meristem, making a notch of great depth, and the edges of the coleoptile rose along the periphery of the meristem, As a result, these events were conducive to the bilateral symmetry of the embryo. At 5 DAA, the coleoptile was overarching the apical meristem. At 8 DAA, the meristem was completely enveloped by the coleoptile and the first and second leaves, resulting in the formation of a plumule. The ventral organization of the shoot apical meristem is discussed in relation to somatic embryogenesis of rice as it is currently mentioned.

The effects of electro- and chemical fusion procedures on cell wall formation and cell division of lettuce and soybean protoplasts were studied. Judging from Calcofluor White staining, cell wall formation of both lettuce and soybean protoplasts was stimulated by electro-fusion procedure 3 h after application. On the other hand, the cell wall formation of lettuce protoplasts was not affected by the chemical fusion treatment using polyethylene glycol (PEG). However, cell wall formation was inhibited with a very dilute concentration of PEG when lettuce protoplasts were mixed with PEG for 3 h followed by Calcofluor White staining. The enhancement of cell division of soybean protoplasts was observed by the electro-fusion treatment 3 days of culture. By transmission electron microscopy, it was also confirmed that the cell wall was detected on the surface of the electrofusion-treated protoplasts 1 day after culture. A cell wall was not observed on the surface of nontreated protoplasts. Moreover, the endoplasmic reticulum in the treated soybean protoplasts increased compared with nontreated ones.

Both abscisic acid and high osmoticum influence (somatic) embryogenesis in plants. However, the specific effects of these bioregulators on general aspects of embryogenesis and storage reserve accumulation, in particular, remain quite unclear. Our results showed that abscisic acid and sorbitol could, depending upon their concentrations, act in ways that are either beneficial or detrimental to the frequency of somatic embryogenesis in sugarcane ( Saccharum officinarum L.) callus cultures. Complementary studies of sectioned scutellar tissues, using transmission electron microscopy were conducted to examine the in situ localization of storage reserves in cells of somatic embryos treated with : (i) 10-5 M ABA ; (ii) 9% sorbitol ; (iii) 10-5 M ABA and 9% sorbitol ; and (iv) untreated embryos (control) for 14 days. They indicated that these substances elicited storage reserve accumulation differently. Whilst ABA-treated embryos allocated significantly more of their cell volumes to protein bodies with no visible amyloplasts, sorbitol-treated embryos allocated more cell volumes to prominent starch grains and numerous lipid bodies. It is concluded that the specific control mechanism(s) of influence due to these compounds is probably different, as reflected in the varying patterns of amyloplasts (or starch grains), protein- and lipid bodies visualized in the scutellar cells.

A histological and ultrastructural study of somatic embryogenesis in coffee embryos was made by light microscopy and transmission electron microscopy. A comparative study on somatic and zygotic embryos of coffee was also performed. The cells in the suspensor region of somatic embryos were filled with large-and small-sized granules containing polyphenol substances stainable in black. The granule size increased until the elongated-embryo stage and thereafter it decreased with embryo development. Such granules were very small in cotyledonary embryo cells. The continuous and discontinuous cuticular layers were detected on both the somatic, globular embryos and the zygotic proembryos by treatment of iodine/potassium iodide-sulfuric acid-silver proteinate. This preparation also permitted the identification of the deposition of fine silver particles inside the polyphenol granules. In addition the plastids were filled with several starch grains of large and small sizes. Similarly, cells of the zygotic proembryos and young embryos were also filled with polyphenol granules and plastids containing starch grains. It is interesting to note that mucilaginous materials cover the surface of both the embryogenic callus and the suspensor region of somatic embryos and are also present in the intercellular spaces of these tissues. These materials were also covering zygotic embryos situated in the embryo cavity of seeds. These observations led to the conclusion that the somatic and zygotic embryos have several similarities at the ultrastructural level.

Protoplasts of soybean (Glycine max (L.) Merr.) were isolated from suspension cultures and morphological changes during culture were observed using transmission electron microscopy. The regeneration of cell walls on the surface of the protoplasts was accompanied by the release of smooth vesicles from cytoplasm to the outside plasma membrane. The vesicles were presumed to carry some wall materials from cytoplasm for cell wall formation. With the elapse of time, the wall materials were gradually deposited and a complex cell wall layer was formed 2-3 d after culture. The fine structure of cellulose microfibrils was clearly observed about 3 d after culture using a polysaccharide staining procedure. During the cell wall formation, numerous smooth vesicles were always found between the plasma membrane and newly synthesized cell wall. Nuclear division and subsequent cytokinesis were observed about 1 d after culture. From the results obtained by the present and previous researches, it may be concluded that cell wall formation usually starts earlier than cell division and then both reactions proceed concurrently and that smooth vesicles play an important role in cell wall formation.

Cell ultrastructure was examined in rice embryos at the developmental stage between 42 and 72 HAA (hours after anthesis). Until 48 HAA, the cells gradually became cytoplasm-rich with a slight increase in the volume of the embryo exhibiting a globular shape. The globular embryo consisted of two parts : an embryo proper which was spherical in shape and a suspensor situated toward the micropylar end. From 48 to 66 HAA, cells of the embryo proper attained a very meristematic profile with vacuoles becoming small and few in number. On the other hand, in the suspensor region, cells and their vacuoles were larger than those of the embryo proper. Until 66 HAA, the absence of cuticular layer on the surface of globular embryo was ascertained since no materials reactive to the I2KI-H2SO4-AgP method were observed. At 72 HAA, however, a stable layer of cuticle began to appear on the surface of the embryo, extending from the ventral side and gradually covering the entire embryonal surface. Simultaneously, cell differentiation occurred in the internal tissues accompanied by ultrastructural changes. It was concluded from these results that the initiation of a cuticular layer is one of the first cytological events that lead rice embryos to organogenesis.

Embryogenic callus formation and growth of somatic embryos of sugarcane ( Saccharum officinarum L.) were followed by electron microscopy in order to investigate the correlation between the morphological sequence of events and some ultrastructural aspects of storage reserve accumulation. Observations of the differentiation symptoms showed that the embryos pass through distinct morphological stages, superficially reminiscent of those in grass zygotic embryos; with structural bipolar embryonic orientation showing distinctively clear embryo axes and scutellum. Histochemical and ultrastructural examinations revealed that both tissues accumulated significant amounts of the major storage reserves which were sequestered in membrane-bound units, though some differences in storage protein accumulation were observed between them at the later stages of embryo development. Taken together, the developmental and ultrastructural implications of these results are of interest since they show that sugarcane somatic embryos can recapitulate events in zygotic embryos and may, probably, have significance in their utility in synthetic seed technology.

Ultrastructural observations were undertaken to study the viability of fusion products by electrofusing the protoplasts from the mesophyll cells of pansy leaves and the cultured callus cells of wild viola petioles. The result showed that the fusion product was formed between the two different protoplasts and two distinct regions were observed in its cytoplasm ; one region contained chloroplasts from pansy mesophyll and the other, plastids from wild viola callus. In other cases, however, the fusion product was seen which might be derived from merely the wild viola protoplasts because of the lack of well-developed chloroplasts. Also, other cellular components, such as nuclei, endoplasmic reticula, small vesicles and osmiophilic granules, were clearly found in the fusion products. Further, the fate of fused protoplasts is discussed on the basis of the ultrastructural features.

Up to 12 h after anthesis (HAA), the first cell division in rice zygotes occurred with the formation of a slightly oblique transverse wall, causing the partition of the zygote into an apical cell and a basal cell. At 18 HAA, the partition in the apical cell was brought about by the presence of a longitudinal wall and in the basal cell by a transverse wall, which resulted in the formation of a four-celled embyro. At 24 HAA, the middle cell of the four-celled embryo was partitioned by a longitudinal wall, and the basal cell was again partitioned by a slightly oblique transverse wall. At 30 HAA, the apical and middle regions had four cells each with the further formation of longitudinal walls, so that the embryo became ten-celled. After that, cell division was directed at random. Well-developed cytoplasm existed around nuclei in proembryo cells. The cup-shaped plastids containing starch grains, the cup-shaped mitochondria, and the stratiform type of rough endoplasmic reticula were frequently observed. Many vacuoles were seen in the peripheral cytoplasm. The basal cells were more vacuolated than the cells at the upper side of the proembryo. At 18 HAA, the persistent synergid completely degenerated, involving electron dense materials. The endosperm became cytoplasm rich around the proembryo, especially after the embryo had started to increase in size.

Protoplasts were enzymatically isolated from soybean suspension cells and were cultured in a liquid medium. The reappearance of cell wall at the surface of naked protoplasts was studied by fluorescence and scanning electron microscopy. Using fluorescence microscopy (Calcofluor White staining method), the cultured protoplasts regenerated new cell walls only a few minutes after isolation and protoplasts were completely covered with new wall material after 9-7 12 h. The first division of the protoplasts was found 15 h after culture and often observed after 1-2 d culture. These regenerated cell walls could be digested with cellulase but not pectinase. By scanning electron microscopy, the wall materials deposited on the surface of protoplast were observed. After 9 h culture, short fibrils attached to projections on the plasma membrane. After that, long fibrils were formed and spread over the surface. After 1 d culture, the fibrous materials which had already deposited on the surface transformed to complex wall structure. The dividing protoplast was also observed after 12 h culture.

Development of somatic embryos from coffee leaf explants has been studied in detail by scanning electron microscopy (SEM). Friable embryogenic callus is formed by single spherical cell aggregates of about 20μm diameter. They are the putative initial cells of somatic embryos or embryo mother cells. The fine structure of somatic embryos was clearly identified during the following sequential developmental stages : single spherical cell, early globular shape with conspicuous suspensor, typical globular shape, elongated shape, early heart shape, typical heart shape, torpedo shape, and finally cotyledonary stage embryo with well-developed suspensor. The high frequency of somatic embryos was found in callus tissues derived from mature leaves under a two-step culture method. Low frequency somatic embryogenesis was found on the leaf edges associated with very small callus formation. Low frequency somatic embryos were observed only in young leaves under the one-step culture method. In this SEM investigation, it is demonstrated that the sequential events leading to coffee somatic embryogenesis arise through one unicellular pathway derived from coffee somatic embryo mother cells. A schematic representation of the sequential stages of coffee somatic embryo development is provided.

Callus growth parameters such as induction frequency, fresh weight and plant regeneration potentials of ten genotypes of African rice ( Oryza glaberrima ) on Murashige and Skoog (MS) basal medium supplemented with organic nutrients and different levels of two auxin sources as well as the morphological pathway of development leading to plantlet regeneration were studied by scanning electron microscopy. Each of the two synthetic auxins tested: 2,4-dichlorophenoxyacetic acid (2,4-D) and 3,6-dichloro- o -anisic acid (dicamba) was found to have appreciably different effects on the callusing ability of the genotypes at different concentrations. Generally, dicamba was observed to stimulate the formation of a mainly greenish, hard and nodular compact callus phenotype which yielded a higher plant regeneration frequency than 2,4-D in most of the genotypes. Sequential electron microscopy of the callus during growth and differentiation demonstrated that different developmental features were present on the surfaces before the eventual initiation of shoot and root primordia. On the basis of the structural data, the histogenetic event is best ascribed to the non-synchronized, independent growth of callus leading to organogenesis of shoot and root meristems.

The scanning electron microscopy-light microscopy method (SEM-LM), routinely prepared SEM specimens can be reprocessed for light microscopy (LM), is presented. In this method, specimens were embedded with the water-soluble resin glycol methacrylate (GMA), which offers a broad histological stainability for LM, so the present method is applicable to several types of specimens.

The whole surface of rice embryo was continuously covered with a cuticular layer until 4 days after anthesis. The cuticular layer became intermittent at only the abaxial side of scutellum at 5 days and disappeared completely up at 7 days. By the extinction of this cuticular layer, it was possible to distinguish scutellar epithelial cells from epidermal cells on the other side of the embryo. Therefore, scutellar epithelial cells began to establish a peculiar nature from about 5 days. Epithelial cells, in this time, were still rich in cytoplasm and showed anticlinal divisions. At the end of the anticlinal divisions, epithelial cells gradually altered to an elongated profile in shape. Tiny ingrowths of cell walls were often observed at the surface of cell walls. The walls of epithelial cells were lengthened at a right angle to the outer surface and thickened about half the length inward from the surface, showing unique characteristic of scutellar epithelial cells. Thereafter, the number of lipid bodies and amyloplasts increased in the cells and vacuoles accumulated their contents and transformed to protein bodies. As the embryo developed into the dormant stage, lipid bodies were displaced near the cell walls. By using freeze-substitution method, it was noticed that membrane systems are smooth and the inside of mitochondria or plastids is more electron-dense in comparison with chemical fixation. The presence of tiny ingrowth and thickening of cell walls was also observed by freeze-substitution method.

Rice coleoptile differentiated at ventral side of embryo and covered a shoot apex during 3 days to 5 days after anthesis. Then the epiblast and lateral scale initiated and surrounded the coleoptile. Simultaneously surface area of scutellum expanded, and the axis of plumule and radicle increased in diameter, and finally ventral scale differentiated as a scutellar appendix. During rice seed germination, lateral scale, epiblast, and coleorhiza grew and emerged from a rent of the seed coat. Then they had many hairs in the surface and the coleoptile appeared from the opened lateral scale. Seminal root pierced coleorhiza and emerged geotropically from surface of coleorhiza in touch with agar surface. Furthermore, a technical term of lateral scale is discussed.

The present study is intended to analyze the early response of protoplasts isolated from soybean suspension cells to electrofusion. It was observed by light microscopy that protoplasts formed a pearl chain-like structure after AC application, started to fuse after. DC pulse application and became round-shaped cells within five to fifteen minutes. Electron micrographs showed that after giving DC electric pulses, the originally smooth surface of some protoplasts became rugged. This structural change was observed mainly on the contact areas or opposite sides of two protoplasts. Then, fusion started at the narrow areas on the surface membrane of protoplasts in contact with one other. Next the fusion area increased and the cell components mixed slowly. Finally, the fusion bodies became round. Based on the above results we believe that the fusion process consists of five steps : adhesion of protoplasts, deformation of protoplast surfaces, fusion in narrow areas, progress of fusion between protoplasts and formation of round-shaped fusion bodies.

Mature seeds of five cultivars of Asian rice (Oryza sativa L.), a variety of African rice (Oryza glaberrima Steud.) and two varieties of wild rice (Oryza perennis Moench and Oryza latifolia Desv.), cultured on 2, 4-D containing nutrient media showed rapid proliferation of the scutellum and high efficiency in morphogenic callus formation. Varying concentrations of sucrose were found to have different effects on callus growth and plant regeneration. High sucrose concentrations in the callus induction medium promoted callus formation and subsequent organogenesis. Callus produced by the cultivars and varieties of wild rice germinated in vitro and produced plantlets. Morphological evidence is provided to indicate that plant regeneration occurred through organogenesis. Aspects of interspecific relations are also discussed.

The surface feature on the initiation and development of callus, derived from coffee (Coffea arabica L. var. typica) leaf explants, was examined by scanning electron microscope (SENI). After about two weeks of culture, callus emerged mainly from cut leaf edges. After about 45 days, these callus masses turned into soft and translucent tissues which were like an assembly of numerous threads at a stereomicroscopical view. Ultrastructural observations by SENI revealed that callus proliferation mainly occurred from mesophyll cells near vascular bundles of cut leaf explants. A membranous layer covering the callus surface was broken by some ruptures at several places, and superficial cells appeared underneath the fragmented membrane. Furthermore, interesting morphology of the callus surface was revealed: spherical cells, elongated cells, and cylindrically elongated or curled cells were present.

A light and transmission electron microscope study was made on the disposition of an egg cell and two synergids in Oryza sativa ovules. After anthesis, one of the synergids is rather persistent while the other is degenerating with collapse of the plasma membrane and penetration of ground plasma into the space between the plasma membranes of the egg cell and the central cell. Spherical vesicles in degenerating synergids closely resemble polysaccharide particles in intact pollen grains. Singular cisternae in the central cells tightly associate with the plasma membrane which borders on the degenerating synergids. There are osmiophilic deposits in the narrow interspace between the plasma membranes of the egg cell and the persistent synergid or the central cell. The egg cell has a nucleus with two nucleoli. The persistent synergid has a malformed nucleus forced on a pocket portion of central cell. The portion is occupied by a large number of cell wall ingrowths. It is postulated from the cyto-histological features of ovaries that nutrition into the egg cells is supplied through transmembranal, symplastic and apoplastic routes.

Plantlets were regenerated from 5-year subcultured compact callus derived from petiole tissues of wild viola (Viola patrinii DC.) but not from 5-year subcultured friable callus. Regeneration occurred most efficiently on medium that contained two-fold diluted basal salts of Murashige and Skoog's (MS) medium, 5 × 10(-6) M 1-naphthaleneacetic acid and 10(-6) M kinetin. The effect of dilution of MS basal salts could also be achieved solely by two-fold dilution of the potassium dihydrogen phosphate in the mixture.The present study revealed that dilution of MS basal salts, in particular of potassium dihydrogen phosphate, was important for the regeneration of wild viola. Moreover, although the callus had been subcultured for 5 years, regeneration of plantlets from callus was still possible. In addition, scanning electron microscopy revealed that details of the process of plant regeneration from subcultured callus varied with the age and source of callus and differed from that reported in rice.

Protoplasts were isolated from rice callus and lettuce cotyledons. Their fusion process was studied by light and electron microscopy. Lettuce protoplast fusion could be easily produced by either of polyethylene glycol and electric methods. The transformation of fusion bodies during the fusion process was more easily observed by using the electric method than chemical one. After electric pulse application, the fusion of lettuce protoplasts was finished within about one hr, judging from their externals. On the other hand, the fusion of rice protoplasts occurred in a very short time after electric pulse application. Fusion was performed between lettuce protoplasts and rice protoplasts. Rice protoplasts contain no chloroplasts and lettuce protoplasts contain them. Therefore, both protoplasts could be easily distinguished under a light microscope. When the fusion between rice and lettuce protoplasts was induced by electric pulse application, chloroplasts in lettuce protoplasts moved into rice protoplasts within about one hr. Electron micrographs showed that the components from both protoplasts was located separately in the fusion bodies. Big rice fusion bodies produced by fusion of rice protoplasts contained some round-shaped particles. Their size was almost the same as that of the original protoplasts. These results indicate that it take a long time until the cytoplasm of rice protoplasts mix with the cytoplasm of lettuce protoplasts in the fusion body.

Callus induction, subculture and shoot formation were investigated using barnyardgrass seeds. Relatively high levels of 2, 4-D (2×10-5M or 4×10-5M) were required for the callus induction. The callus cultures were white or yellow in color and often altered black brown under the prolonged cultivation. Hetero-geneous tissues of the cultures were remarkable in comparison with rice cultures: The higher kinetin were added to the medium, the more shoots were formed from the callus cultures. For electron microscopy the cells of callus tissues were fixed by glutaraldehyde and osmium tetroxide, and embedded in Epon 812. Thin sections were stained with uranyl acetate and lead citrate and examined with Hitachi HU-12 A microscope. Many of osmiophilic granules were seen in the proplastids. Secondary vacuoles containing cyto-membranes were often observed in the callus cells. In the green regions of cultures, amyloplasts containing the large granules of starch and chloroplasts were seen. The peripheral reticulum was indicated in immature chloroplasts. It was also confirmed that mature chloroplasts have lamellar structure and multilayered grana and chloroplast-DNA filaments are occurred in some regions of the matrix having low electron density.

The regeneration potential of shoot tip, stem, leaf, cotyledon and root explants of two papaya cultivars (Carica papaya cv. 'Solo' and cv. 'Sunrise') were studed. Callus induction of these two cultivars of papaya showed that the shoot tips and stems are most suitable for forming callus, while leaves, cotyledons and roots are comparatively difficult to induce callus. Callus induction also varied with the varities. Somatic embryogenesis was obtained from 3-month-old root cultures. A medium containing half strength of MS inorganic salts, 160 mg/l adenine sulfate, 1.0 mg/1 NAA, 0.5 mg/1 kinetin and 1.0 mg/1 GA3 was optimal for embryogenesis. The callus maintained high regenerative capacity after two years of culture on this medium. Plants derived from somatic embryos were obtained under green-house conditions.

Light quality and quantity of the environment, where lettuce seedlings were grown, affected the mesophyll tissue and the shoot formation in cotyledons excised and cultured in vitro. When cultured on MS medium for 5 days, the cotyledonary tissues of the seedlings were poorly developed under an illumination level of 150 lx with fluorescent light but well-developed both under 5, 000 and 10, 000 lx, The mesophyll cells of cotyledons were rich in cytoplasm when excised from the seedling grown under BOC lamp or 150 lx of fluorescent light but vacuolated under 5, 000 and 10, 000 lx. Frequecy of adventitious buds on the cotyledons grown under high-level light intensity was less than low-level intensity. In addition, the adventitious bud formation decreased as the age of the cotyledons advanced. This trend was clear under the high light intensity.

The experiments were performed to clarify the tissues from which callus is induced in rice seed. Dehusked seeds (var. Aichiasahi) were inoculated aseptically on Maeda's medium containing 3% sugar, 0.3% casein hydrolysate and 10-5 M 2, 4-dichlorophenoxy acetic acid (2, 4-D), and cultured for 30 days at 30°C under light condition. Morphological and histological changes of the tissues during culture period were as follows. The growths of the plumula and radicle were suppressed, although the scutellum and mesocotyl tissues proliferated abnormally. Tissue enlargement occurred in the scutellum, in which the parenchyma cells expanded and the epithelium cells divided 3 or 4 times for the first 7 days. After 20 days of culture epithelium bigan rapid cell proliferation to produce callus tissue. The other hand, in the epithelium on the medium without 2, 4-D a cell division occurred once. In the mesocotyl, cell divisions bigan at the outermost layer of the central cylinder leading to that of the radicle for 2 days of culture and the cell divisions expanded to the radicle and the vascular tissue of the scutellum. After that they proliferated consistently during culture period and produced callus tissue. The fresh weight of an embryo became 225 fold at the end of culture. On the contrary, a vascular system fully developed in the mesocotyl of the control seedling lacking 2, 4-D which grew normally. The endosperm digestion of callus forming seeds delayed considerably since the some dehydrogenase activities in thc epithelium cells would fail. The morphological changes accompanying with callus formation occurred even when the seeds removed from callus inducing medium containing 2, 4-D to the medium lacking 2, 4-D after 2 or 3 days of culture, suggesting that irreversible change of the tissues had already occurred for this period. Mesocotyl, scutellum and radicle dissected from embryos of dry seeds were cultured under above condition, but they did not produce callus tissue. Dissected plumula and the tissue having both plumula and mesocotyl produced callus tissue. This suggests that plumula would play physiologicaly important roles and that callus formation in mesocotyl and scutellum tissues in a seed would occure under the physiological interactions between plumula and these tissues. Furthermore, when 2, 4-D was decreased to 3×10-5 M the outermost layer of the central cylinder in mesocotyl formed stunted primordium of lateral roots, showing that callus tissue of mesocotyl is formed by abnormal proliferation of the cells which should form lateral roots.

1. Callus tissues were induced by various concentrations of 2, 4-dichlorophenoxyacetic acid (2, 4-D) from different organs, roots, mesocotyls, internodes, nodes, coleoptiles, leaf sheaths, and epidermal cells of scutellum of Oryza sativa and roots, leaves and stems of Echinochloa crus-galli, Nicotiana tabacum, and Vicia faba. 2. Callus induction is more remarkable in some organs of young rice seedlings than old ones with lower level of 2, 4-D. 3. Different concentrations of 2, 4-D are necessary for callus induction from different organs and diverse species. Tissues and organs in rice seeds, from which callus is able to be induced, are strikingly varied by different concentrations of auxin. 4. The concentration of auxin for callus induction is dependent on a sort of auxins used; IAA, NAA, or 2, 4-D. The difference of auxin concentration for the callus induction corresponds with the difference of auxin activity of the substances used. 5. Effect of kinetin on callus induction in rice seeds is not only undistinct but also repressive. 6. Thiamine is required for growth of rice cultures but it is not essential for callus induction in rice seeds. 7. It is discussed on the effects of the cutting of seedlings on callus induction in rice. 8. The tissues from which the callus is originated in various concentrations of 2, 4-D is histologically investigated on rice seeds and segments excised from the seedlings. Anatomical studies indicated that the initiation of callus in various organs resembled to the initiation of the adventitious root, except for epidermal cells of scutellum. 9. The organ differentiation in rice callus tissues is controled with various concentrations of 2, 4-D. Namely, callus tissues without root-like structures, callus tissues with root-like structures, abnormal roots and normal roots are formed as the concentration decreases.

Developmental processes of shoot formation in rice callus tissues of var. Aichi asahi and Te-Tep grown in the organ forming medium were investigated in detail. At the same time, the changes in various hydrolytic enzyme activities (α-amylase, RNase; catalase, acid phosphatase) and α-amylase isozyme patterns in these processes were studied and compared with the some biochemical changes in rice seedlings. For this purposes, the callus tissues with green or white shoot protuberances were divided into organ-forming regions and non-organ-forming callus parts. (1) The clear cut results on organogenesis in callus tissues were obtained by adding the casein hydrolysate (4 g/1) and 5×10-5 M kinetin to Maeda's medium containing 10-7 M 2, 4-D and by using the 3 passage callus tissues, in light (3, 000 lux) conditions of 25°C instead of 30°C. Such conditions as above mentioned inhibited early growth of callus parts showing organogenesis. The callus tissue in these conditions was designated as K-callus tissues. (2) In K-callus tissue, organ-forming regions and shoot primordia were observed after 6 days of callus innoculation. Also, various hydrolytic enzyme activities during these periods were rather inhibited in comparison with control callus tissue in the 10-5 M 2, 4-D medium without kinetin (C-callus). (3) Shoot formation began to start gradually after a week and became vigorous from 10 to 14 days in culture. But root formation was slight at this stage. As the shoots were formed, RNase and α-amylase activities in shooting regions increased rapidly. α-Amylase activity was maximum at 10 day-old. At the 10th day, RNase and (α-amylase activities were about 7- and 10-times as much as that of C-callus tissue, respectively (fig. 12, table 2). These data suggest that histological and biochemical "lag phase" in organogenesis present about one week after callus innoculation. (4) In the callus tissue grown in the GA3-treated (5×10 M) organ-forming medium (KGcallus tissue), organogenesis and its related enzyme activities were strikingly decreased (fig. 13). (5) In the organ forming medium containing yeast extract (5 g/1), the shoot or root primordia of Te-Tep callus tissue were observed after about two week. The shoot primordia were initiated exogenously and root ones endogenously, and both primordia were formed independently to each other. The shoot primordia were circularly surrounded by a large number of vascular bundles, but the root ones were not. Their singularities were discussed. (6) From the observations on α-amylase banding patterns, the results were obtained that the activities of α-amylase "g" and "e" conspicuously increased and a new isozyme "1" was induced with organ developments in K-callus tissue and in the organ forming regions of KG-callus tissue. (7) Several hydrolytic enzyme activities in rice Seedlings of 2 to 4 day-old were studied to compare the changes in developmental processes in redifferentiation of shoot. Enzyme activities were lower than those of C-callus (10 day-old) or shoot-initiating regions of K-callus tissue except catalase. Only one isozyme of α-amylase which was considered to correspond with amylase "a" in callus tissue was isolated from rice seedlings. From the results mentioned above, the intimate relations between histological and gross morphological aspects and some biochemical changes in redifferentiation processes of rice callus tissues were suggested. The role of hydrolytic enzymes and vascular systems surrounding circularly shoot primordia in these processes were discussed.

The effects of oxadiazon; 5-t-butyl-3-(2, 4-dichloro-5-isopropoxyphenyl) 1, 3, 4-oxadiazoline-2-one, which represents the herbicidal action only under the light condition, on the ultrastructure of chloroplasts of barnyardgrass (Echinochloa crus-galli) was studied electronmicroscopically. When the etiolated seedlings, grown for three days in oxadiazon solution under dark condition, were exposed to light, greening and elongation of the leaves were inhibited markedly. Most of leaf tissues were considerably distorted by the action of oxadiazon. Especially disintegration of vascular bundle was remarkable. In chloroplasts of untreated leaf cells, grana was formed and starch grains were also found in some case. However, in the case of herbicide treated plants, chloroplast development was suppresscd. These chloroplast were characterized by having prolamellar bodies and circular lamella structure. However, even in cells whose nucleus and cytoplasm were distinctly affected, rupture of the chloroplast with disintegration of envelop membrane was not observed and their shape were kept their original form. It is proposed from the above mentioned results that oxadiazon influenccs in not only chloroplasts but also nucleus and cytoplasm in spite of the well known action of the substance which closely related with the light exposure.