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水稲の湛水直播栽培における耐ころび型倒伏性を改善し, 生産性の安定化に役立つ水管理法を明らかにする目的で, 落水管理の処理回数, 期間等の異なる水管理条件で栽培した直播水稲を対象に, 押し倒し抵抗, 倒伏程度, 収量および品質を比較調査した. 押し倒し抵抗は, 落水管理の処理回数が多く, 処理期間の長い区でより高い値を示した. こうした落水管理の押し倒し抵抗に対する向上効果は, 播種深度が異なってもほぼ同様で, 播種深度が1mm以下の表面播種区においても落水管理を行うことによって押し倒し抵抗が高まる傾向が認められた. 地表面に播種された散播栽培における倒伏は常時湛水区でもっとも顕著に発生し, 以下, 落水1回区, 2回区と落水管理の処理回数が多く, 処理期間のより長い区で倒伏程度が軽減される傾向がみられた. 収量については, 倒伏の発生程度の著しかった常時湛水区では収量の低下がみられたが, 7日程度の落水管理を3回まで実施しても収量や品質に悪影響は認められなかった.

Intergeneric somatic hybrid calli were obtained through electrofusion between protoplasts of Celosia cristata L. (C3 species) cell suspension and Amaranthus tricolor L. (C4 species) cotyledon. The highest binary fusion frequency, 4∼5%, was attained when the protoplasts were suspended in fusion solution containing 0.4 M sorbitol and 2.0 mM CaCl2, aligned by an AC field (200 V/cm, 0.5 MHz) for 30 s and fused by a single DC square pulse at 1.6 kV/cm for 40 μs. Fusion products were identified by characteristics of both parents in the hybrid cells : they possessed green chloroplasts or pink plastids from cotyledonary protoplasts as well as a cytoplasm-rich colorless sector derived from cell suspension protoplasts. The occurrence of two types of calli, compact and friable, obtained after culturing on proliferation medium lead to the isolation of putative hybrid calli without any artificial selection because C. cristata protoplasts regenerated only friable calli and A. tricolor protoplasts did not produce calli. A total of 14 lines of putative hybrid compact calli were obtained by screening on the basis of callus morphology. An isozyme analysis of acid phosphatase confirmed the hybridity of 14 lines of compact calli. Callus morphology was also studied by scanning electron microscopy.

Mesophyll protoplasts of mung bean (Vigna radiata L. Wilczek) were electrofused with cell suspension-derived protoplasts of adzuki bean (V. angularis Ohwi & Ohashi) and subsequently cultured for several days. The structural characteristics of fusion products and unfused protoplasts of parental species were examined using light and transmission electron microscopes. Fusion product was distinguishable from the parental species due to the concurrent existence of chloroplasts originated from mung bean and proplastids from adzuki bean. Heterokaryon performed a mixture of cytoplasms between fused parentals. However, chloroplasts from mung bean and proplastids and ER fragments derived from adzuki bean were still encountered on their initial sides. Chloroplasts became a little round in shape due to application of electric field. Electric stimulation also caused the fusion between organelles. The fusion product started to divide after 5 days in culture. Some of the dividing cells demonstrated unequal segregation of chloroplasts in the daughter cells.

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.

Ultrastructural changes in chloroplasts of peanut (Arachis hypogaea L. var. Lokal) seedling mesophyll cells and mesophyll protoplasts treated with electric fields were investigated. Generally, chloroplast profiles in mesophyll cells were biconvex, whereas those in mesophyll protoplasts were oval and round and some were divided. When the protoplasts were treated with electric fields, some chloroplasts were paired. Some of these seemed to result from division and others from fusion. This electric field treatment may stimulate chloroplast division and fusion, related to the activation of protoplasts which has been known to occur by the same treatment. There are two types of division, i.e. partition and fission. The division by partition was usually observed by invagination of the inner membrane of the chloroplast envelope. The fission type of division was recognized by a dumbbell-shaped profile and electron dense material was usually observed in the neck region. Some chloroplast profiles seemed to be produced by fusion of two chloroplasts, because the two parts had a common envelope and two thylakoid systems at the fusion area were disharmonious.

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 were successfully isolated from mungbean leaves (Vigna radiata L. Wilczek) using an enzyme solution containing 4% Meicelase P-1, 1% Macerozyme R-10, 1% potassium dextran sulphate and 0.5 M mannitol. Approximately 4.1×107 protoplasts per gram fresh weight were released after digestion with an enzyme for 4 hours in the dark on a shaker. The cultured protoplasts started to divide 5 days after culture followed by sustained division, and a microcolony was produced on day 15 of culture. Microcalli appeared on the medium surface a month after culture and were large enough for transfer to a callus induction medium after 1.5 month of culture. Having transferred to the regeneration media, green and compact calli were developed in MS medium supplemented with NAA and BA. Globular-like calli appeared in the MS basal medium or in the mixture of MS salts and B5 vitamin supplemented with 2, 4-D. Root formation then occurred from this calli.

High yields of viable protoplasts were obtained from leaf mesophyll of 10-day-old peanut seedlings (Arachis hypogaea L. var. Lokal). An enzyme solution containing 1% Cellulase YC, 2% Meicelase P-1, 0.15% Pectolyase Y-23 and 0.5% Macerozyme R-10 was found to be the best for protoplast isolation. The protoplasts were then cultured in liquid or in agarose-gel modified KM8P medium. Cultured protoplasts divided 2 days after culture. In solidified media, protoplasts divided continually and formed many colonies 1 month after culture. The best viability and colony formation was obtained in a culture medium solidified with 1.2% agarose.

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.

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.

High yield of protoplast was obtained from the green callus tissue of finger millet at one week after subculture on MS medium with 0.2 mgl-1 2, 4-D and 0.1 mgl-1 kinetin. The protoplasts were released using the enzyme solution containing 2% Cellulase YC and 0.2% Pectolyase Y-23. About 75% of the protoplasts cultured in the MS liquid medium with 0.4 mgl-1 2, 4-D and 0.1 mgl-1 kinetin supplemented with 5% coconut water were started to show regeneration of the cell wall within 12 h. Cell division was observed within 24 h after culture. The protoplasts that underwent division were about 15%. The sustained cell division could lead to microcolony formation in 4 d after culture. However, further growth did not occur and viability gradually decreased during culture.

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.

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.