Yoji Takeoka

Effects of sodium chloride on foliar ultrastructure of sweet potato (Ipomoea batatas Lam.) plantlets grown under light and dark conditions in vitro

Summary The effects of sodium chloride on foliar ultrastructure of sweet potato plantlets grown under light and dark conditions in vitro were examined. Ultrastructural changes induced by salt stress under light conditions occurred in the following order: (1) vacuolation, development and partial swelling of ER; (2) decrease in mitochondrial cristae and swelling of mitochondria, with an increase of the vesicles released from the Golgi stacks; (3) vesiculation and fragmentation of tonoplast; and (4) degradation of cytoplasm by the mixture of cytoplasmic and vacuolar matrices. In the mesophyll, thylakoid membranes of chloroplasts were swollen and most were lost following severe salt stress. Under dark condition, however, inner membranes of etioplasts maintained their ultrastructural integrity. The present study suggests that degradation of cytoplasm and cell organelles, except thylakoid membranes of chloroplast, occurred as a result of light-independent salt stress, and degradation of thylakoid membranes of chloroplast in the mesophyll occurred as a result of salt-induced oxidative stress.

Effects of Exogenous Glycinebetaine on Growth and Ultrastructure of Salt-Stressed Rice Seedlings (Oryza sativa L.)

Abstract The effects of exogenously applied glycinebetaine on the salt-stress-induced inhibition of growth and ultrastructural damages in rice seedlings were investigated. Glycinebetaine was not effective in alleviating the NaCl-induced inhibition of root growth and rather enhanced the NaCl-induced inhibition. However, it was found to alleviate the inhibition of shoot growth induced by NaCl stress. Concentrations of Na were higher in salt-stressed plants than in unstressed plants. Stressed plants receiving glycinebetaine had a significantly lower Na and higher K concentrations in the shoots than the plants grown without application of glycinebetaine. Salinity induced ultrastructural damages in leaf such as swelling of thylakoids, disintegration of grana stacking and intergranal lamellae and destruction of mitochondria (deficiency of cristae, swelling and vacuolation). Such damages were largely prevented by pretreatment with glycinebetaine resulting in greening of the plants. In roots, the epidermis, cortex and root cap were more sensitive to salt stress than the meristem and stele. The most frequently observed ultrastructural alteration due to NaCl salinity was the formation of many large vacuoles in the root tip and root cap cells. The number of mitochondria was increased and they were aggregated in the cytoplasm of the root tip and root cap cells by treatment with NaCl or NaCl plus glycinebetaine. Glycinebetaine could not prevent the NaCl-induced ultrastructural damages in root cells. The effects of glycinebetaine to mitigate the ultrastructural damages in the chloroplast and mitochondria induced by NaCl might be due to the production of many vacuoles in root cells which may act to store Na and decrease its accumulation in the shoot.

Salinity-Induced Ultrastructural Alterations in Leaf Cells of Rice (Oryza sativa L.).

Summary High concentrations of NaCl significantly reduced the fresh and dry weights and lengths of roots and shoots. NaCl exhibited a more rapid effect in water culture than in soil culture. In both water and solid cultures, root growth was suppressed more severely than shoot growth. Electron microscopic studies revealed that NaCl caused swelling of thylakoids, accumulation of starch grains and lipid droplets, distortion of grana stacking, increase in the size and number of plastoglobuli and vesiculation of cellular membrane. Mitochondria became deficient in cristae, swelled and the matrix appeared pale in salt-treated plants as compared with control plants. Disappearance of nucleolus and nuclear chromatin and destruction of vascular tissues were occasionally observed in salt-treated plants.

Distribution of manganese in leaf tissues of manganese accumulator: Acanthopanax sciadophylloides as revealed by Electronprobe X‐Ray Microanalyzer

Abstract The distribution of Mn in leaf tissues of a Mn accumulator: Acanthopanax sciadophlloides was investigated by means of electron‐probe X‐ray microanalysis using a scanning electron microscope in conduction with a cryostage. Unfixed and frozen plant specimens were used in this experiment. The results show that Mn is densely accumulated in the peripheral cells either of leaf, petiolule and petiole tissues. In the leaf tissues, Mn was highly concentrated in epidermis, palisade and spongy parenchyma cells. Considerable low Mn concentration was observed in vascular bundle cells. Distribution pattern of Mn in petiolule and petiole was similar and characteristic. In petiolule and petiole, most of the Mn was accumulated in the cell wall of epidermis, collenchyma and bundle sheath cells and least was in vascular bundle cells.

Pistil hyperplasia in rice spikelets as affected by heat stress

SummarySexual organogenesis in spikelets of rice (Oryza sativa L. cv ‘Kinmaze’) affected by heat stress was investigated using SEM and stereo-microscopy. Of the 243 spikelets dissected, 55.6% developed pistil hyperplasia, i.e., proliferated female organs or tissues, including multiple stigmata and/or ovaries, outgrowth of swollen parenchymatous tissues from inside the ovule, and differentiation of trichomes from ovary epidermis. Conversely, 7% of the spikelets exhibited stamen hypoplasia, represented by a decrease in the number of stamens, and only 3.7% of the spikelets showed hyperplasia, represented by an increase in the number of stamens. The morphological and structural development of the anther was disturbed, and microsporogenesis was inhibited. The shape of the anther was altered, though not perfectly, into the form of an ovary; the lemma and palea changed in form and ultimately resembled each other in shape and size. All of these changes in structure are more or less similar to those that usually result in rice spikelets subjected to cold and other environmental stresses. It was therefore concluded that rice plants show similar responses in sex differentiation in the spikelet under various environmental stresses.

Effects of Salinity Stress on the Seminal Root Tip Ultrastructures of Rice Seedlings (Oryza sativa L.)

Abstract Growth and structural changes in the seminal root tip of rice seedlings (Oryza sativa L. cv. Nipponbare) in response to NaCl salinity were studied. Seedlings were grown in agar medium with 0, 0.1, 0.3, 1.0, 2.0 and 3.0% NaCl(agar culture), and in water with 0, 0.01, 0.03, 0.06 and 0.1% NaCl (water culture). Seedling growth was significantly suppressed by higher concentrations of NaCl. The effect of NaCl appeared faster in water culture than in agar culture. In both agar and water cultures, root growth was markedly suppressed over shoot growth. Under saline conditions, epidermis, cortex and root cap cells appear to be damaged to a greater extent than the meristem and stelar cells. The most notable ultrastructural change in response to salinity was the development and increment of vacuoles, which seem to provide a space for accumulation of excess ions. Many electron dense deposits were observed in the larger vacuoles of the epidermal and cortical cells. Under saline conditions, cell wall thickening of the epidermis, an increase in endoplasmic reticulum, myelin figures, less compact Golgi bodies and inhibited production of Golgi vesicles were also observed.

Improvement of Direct Somatic Embryogenesis in Rice by Selecting the Optimal Developmental Stage of Explant and Applying Desiccation Treatment

Summary By selecting the optimal developmental stage of zygotic embryos used as expiants and applying desiccation treatment, we improved direct somatic embryogenesis in rice scutellum from two cultivars, Nipponbare and Sasanishiki. Zygotic embryos isolated 14-17, 21, 28-30 and 35-40 d after anthesis (DAA) from Nipponbare and 14-17, 18-21, 28-30 and 40-42 DAA from Sasanishiki were cultured on the embryo induction medium (EIM). Then they were transferred to embryo maturation medium (EMM) and germinated on the embryo germination medium (EGM). Only immature zygotic embryo isolated 14-17 DAA from Nipponbare and Sasanishiki could develop somatic embryos that germinated. Explants from embryos at other developmental stages could develop somatic embryos only until the elongating or scutellar stage. They enlarged and formed callus without further development. The EIM and EMM consisted of N6 macronutrients, B5 micronutrients, and B5 vitamins, supplemented with 0.1 g L-1 casein hydrolysate, 1.5 g L-1 proline and 1 g L-1 MES buffer. EGM consisted of MS macro- and micronutrients and MS vitamins without organic supplement. In addition, 2 mg L-1 2,4-D was added to EIM, 1 mg L-1 2,4-D to EMM and 0.01 mg L-1 zeatin to EGM. Developmental processes of somatic embryos derived from the expiants were observed by scanning electron microscopy. Desiccation treatment of maturing somatic embryo was proved to produce fully mature somatic embryos capable of germinating vigorously.

Immunogold Labeling of Rubisco in C4 Plant Leaves for Scanning Electron Microscopy

Abstract Immunogold labeling was applied to scanning electron microscopy to examine the inner structure of leaves of G4 plants, maize and linger millet. Ultrastructural features of the G4 plant leaves were observed on cross sections in the secondary electron imaging mode of the scanning electron microscope. Immunogold localization was well recognized in the backscattered imaging mode at the low accelerating voltage of 5 kV on the specimens treated with tannic acid and Oso4 instead of metal coating. Gold labeling of ribulose 1,5-bisphosphate carboxylase/oxygenase was specifically distributed on the cut surface of bundle sheath chloroplasts of the C4 plants. Gold particles were scarcely observed in mesophyll chloroplasts or other organelles. Freeze-cracking of the leaves was found to be effective to expose the inner structure of the organelles. The secondary and backscattered electron images were also compared under various accelerating voltages.

Genetic Diversity of Regeneration Ability in Anther Culture of Rice(Oryza sativa L.)

abstract Rice (Oryza sativa L.) cv. Asahi has been proved to be a useful genetic resource with a unique regeneration ability in anther culture. Diallel analysis was employed using four varieties showing different responses to four different constituent media. The mode of inheritance in regeneration ability of Asahi changed between dominant and recessive expression depending on medium composition although its regeneration rate was constantly high. On the contrary, the low regeneration ability of Koshihikari behaved consistently in a dominant manner and the high regeneration ability of Tsukinohikari behaved in a recessive manner independent of medium composition. The basic medium used was N6 medium containing 0.1μM 2,4-D, 5μM NAA and 70 g L-1 sucrose and the others were supplemented with 0.1μM BAP, 0.1μM BAP+18 mg L-1 glycine or 0.1μM BAP+18 mg L-1 glycine + and 0.5 g L-1 alanine. The difference of medium composition which affects dominance and recessiveness in regeneration ability of Asahi was affected by the presence or absence of 18 mg L-1 glycine supplemented in the medium.