甜菊组织培养物中叶绿体的超微结构与脱分代

含有叶绿体的甜菊（Ｓｔｅｖｉａｒｅｂａｕｄｉａｎａ）愈伤组织细胞转移至新鲜培养基后,导致光合片层的逐渐减少或消失,最后叶绿体脱分化形成原质体样的结构。超微结构观察表明,光合片层的减少或消失与降解及叶绿体分裂特别是不均等缢缩分裂而致基质组分和类囊体膜稀释有关。这一过程并不完全同步,一些质体含有少量正常的片展而另一些质体含有退化的片层甚至片展结构完全消失。细胞的一个明显特点是细胞器大多聚集在细胞核附近,细胞质增加并向细胞中央伸出细胞质丝。同时可观察到原质体。培养７ｄ后,许多细胞呈分生状态,细胞质富含细胞器,充满了细胞的大部分空间。此时细胞中的质体大多呈原质体状态。在细胞生长的稳定期,质体内膜组织成基质基粒片层,同时质体核糖体增加。文中讨论了高度液泡化细胞脱分化与细胞中叶绿体脱分化的关系。     

Ultrastructural Study of Proplastid Ontogeny in Tobacco Mesophyll Cells in Vitro

Since the discovery of plastid DNA the continuity of plastids has well been established. It is known that in plant cultures a form of plastid can differentiate into others. However, only a little has been made in studing chloroplast dedifferentiation in vitro. In the work present here, we reported on ultrastructural changes of chloroplasts dedifferentiation and the proplastid origin in the mesophyll cells of cultured tobacco leaf explant. Fully expanded leaves of haploid tobacco (cv. Ge Xin No. 1) were cut into pieces of 5–6 mm width. These were inoculated on MS medium supplemented with 1 mg/L 2,4-D and 1 mg/l kinetin. The cultures were maintained at (30±2) ℃ and illuminatied by a bank of fluorescent lamps. For electronmicroseopic investigation, after 0, 1, 2, 3, 6 days of culture small leaf fragments were cut off along the cut edges of the explants. The samples were fixed and processed in the manner as described earlier. The sections were examined with a Hitachi HU-11A or a JEM-100CX electronmicroscope. Electronmicroscopic observation shows that the uncultured mesophyll cells are highly vacuolete, with a thin peripheral layer of cytoplasm in which a nucleus and some chloroplasts and other organelles are found in it. But these cells do not contain proplastids (Fig. l). In the explants cultured for 1 day there are no obviously changes in mesophyll cells, except a few cytoplasmic strands extend from periphery to central vacuole. At 2 days of culture quite obvious changes can be detected. A increase in the amount of cytoplasm becomes apparent and transvacuolar cytoplasmic strands grow up. Following cytoplasmic growth, the nucleus and chloroplasts move away from the peripheral cytoplasm and enter the central vacuolate zone (Fig. 2). At this stage some of mesophyll cells have completed the first cell division. After 3 days of culture numerous mesophyll cells have undergone several divisions and formed multicellular masses. In those subdivided cells a more important change of the chloroplasts is the occurrence of protrusions which we call proplastid buds. This phenomenon has also been named as chloroplast budding. According to observations on a large amount of sections chloroplast budding is a common phenomenon in the dedifferentiating mesophyll cells of tobacco leaf explants. Fig ure 3 exhibits a typical profile of a chloroplast with a proplastid bud. The proplastid buds observed are generally long-oval in shape and 1.0–2.5 μm long and about 0.5–0.7 μm thick. These dimensions agree with those of proplastids in meristematie cells. Inside of proplastids ribosomes and electron opaque areas containing DNA fibrils can be seen (Fig. 3). Near the proplastid buds proplastids can often be found (Fig.5). According to above observations we can conclude that the proplastids in dedifferentiating mesophyll cells originate from the proplastid buds by chloroplast budding. The newly formed proplastids usually surround the nucleus and sometimes undergo equal division to increase their number (Figs.5, 6). There are no inner membranes in the newly formed proplastids except vesicles connected with inner membrane of the envelope (Fig.7). While the proplastids are continuously produced, the chloroplasts themselves are filled with starch and gradually turned to large amyloplasts (Fig.5). On the other hand, a few of chloroplasts can divide into equal parts following the chloroplast budding (Fig.4). Israel and Steward (1967) suggested that when cultured carrot cells developed into plantlets the chloroplasts turned into leucoplastids, chromoplastids or proplastids. However, they did not describe how chloroplast became a proplastid. Several investigators reported that the chloroplasts in the dedifferentiating cells gradually lost their grana and intergranal lamellae and then became eueoplasts or proplastids. But according to our observation in tobacco explants, the initiation of proplastids is due to unequal division of chloroplasts, i.e. “budding fission” as described by Malzan and Miihlethaler in Splachnum ampullaceum. Since the proplastid is an organelle characteristic of meristematie cells, the ontogeny of proplastids and its control mechanism should be very important in studing cell dedifferentiation.     

甜菊叶愈伤组织诱导过程中叶绿体的超微结构变化

观察了甜菊(Stevia rebaudiana Bertoni)叶外植体愈伤组织诱导过程中叶绿体的超微结构变化。结果表明,当叶外植体转移到培养基上培养后,叶绿体的片层结构逐渐退化。在叶绿体发生退化的过程中伴有叶绿体出芽和原质体的形成。推测新产生的原质体来自叶绿体产生的芽状体。而叶绿体本身最后完全解体消失。叶绿体超微结构的这种变化与高度液泡化的叶肉细胞脱分化至分生状态是平行的。随着培养的进行,分生状态的细胞发生液泡化变为薄壁细胞时,在愈伤组织表层的细胞中,质体重新形成片层结构,而内部细胞的质体则充满淀粉粒。     

珊瑚豆果实成熟过程中叶绿体转化为杂色体的研究

珊瑚豆 (Solanum pseudo- capsicum var.diflorum (Vell.) Bitter)果实成熟过程中 ,果实颜色的变化和叶绿素含量降低及类胡萝卜素含量增长相符合。对果实中叶绿体转化为杂色体进行了电镜观察。早期绿色果实的特点是叶绿体具典型的基粒 -基粒间类囊体结构。在黄绿色果实时期叶绿体类囊体系统解体 ,代之以少数非叶绿素的单个类囊体和积累大的嗜锇的质体小球。质体转变为所谓的原质体。这表明叶绿体在果实成熟中的脱分化过程。当果实达到黄色阶段 ,这些质体所含的质体小球开始从中央形成质体小管的结构。最初质体小球中央变为半透明 ,认为是质体累积胡萝卜素的开始。随着质体小球的延长 ,小管从小球中伸出。这些小管围以电子致密的膜 ,中央是半透明的轴心。与此同时 ,在质体基质中出现一系列发育不同阶段的小泡 ,似乎是形成新的质体小球的过程。在成熟的橙色和橙红色果实中的杂色体中只包含无数小管和小的质体小球。质体小管在数量和长度上增长 ,充满成熟的杂色体。无数质体小球分布在小管之间的空间中。成熟杂色体从脱分化的原质体的重建是真正的再分化过程。可以作出结论 ,珊瑚豆果实叶绿体转化为杂色体实质上是一个脱分化和再分化过程     

THE ULTRASTRUCTURAL MORPHOLOGY AND ONTOGENY OF OAT COLEOPTILE PLASTIDS

Structurally similar proplastids occur in the shoot, scutellum, and root of the oat embryo at the start of germination. These proplastids follow several pathways of differentiation, depending on their location within an organ and on previous exposure to light. During the first 24 hr of germination morphologically similar amyloplasts are formed from the preexisting proplastids in most of the cells of the seedling. After about 24 hr in the light, unique chloroplasts begin to develop in a subepidermal ring of small cortical parenchyma cells in the coleoptile and give the organ a pale green color. At 48 and 72 hr the coleoptile chloroplasts and etioplasts are conspicuously different from the corresponding leaf plastids in morphology and ontogeny but contain typical photosynthetic grana and prolamellar bodies. Study of the ontogeny of plastids in the epidermal and nongreening parenchymal regions of dark grown coleoptiles shows that these plastids undergo significant losses in starch content, and some increase of membranes within the plastid, related to the age of the cell. Light has little effect on the structure of these plastids. It is suggested that the ontogeny of all the plastid types of the oat seedling begins with a common precursor—a relatively simple proplastid that is present at the time of germination. Starch grains showing two distinct types of erosion, apparently enzymatic, were observed in oat coleoptile plastids. In one type (grooved appearance) the starch grains are consistently associated with plastid membranes, while in the other type (irregular, spiny appearance) the starch grains are associated with the plastid stroma only. We suggest that there are two enzyme systems for metabolizing starch in oat plastids—one membrane-bound and the other free in the stroma.     

THE EFFECT OF LIGHT INTENSITY AND SUCROSE FEEDING ON THE FINE STRUCTURE IN CHLOROPLASTS AND ON THE CHLOROPHYLL CONTENT OF ETIOLATED LEAVES

Changes in the fine structure of proplastids of etiolated leaves exposed to various conditions of light and darkness for 24 and 48 hours were investigated, and the chlorophyll content of the leaves so treated was determined in vivo. The light treatments were given while the leaves were floated on tap water or on a 0.2 M sucrose solution. Leaves floated on water under low light intensity (2 foot-candles) were low in chlorophyll and contained plastids with concentric rows of vesicles. Transferring the leaves back to darkness resulted in the disappearance of the concentric rigs and re-formation of vesicular centers together with straight rows of vesicles and tubules, evenly spaced throughout the stroma. Chloroplasts of leaves floated on a sucrose solution under low light showed large vesicular centers together with stacks of rows of elongated tubules. The same chloroplast structure was found in leaves floated on a sucrose solution in the dark, after having been exposed to weak light for 24 hours. Chlorophyll content in these leaves was the same as in leaves floated on water under high light intensity, where the chloroplasts had normal grana and lamellae. The effect of the investigated factors on plastid development is discussed.     

Ultrastructural Development of Chloroplasts in Sacred Lotus Embryo Bud under Invisible Light

The present paper reports that the development ultrastructural observations of chloroplasts from sacred lotus (Nelumbo nucifera) embryo buds under invisible light. Embryo bud of sacred lotus is enclosed by three layers of thick integument (pericap, seed coat and thick fleshy cotyledons). During the period of the formation of embryo bud, it remained in dark condition, but turned from pale yellow to bluish-green. It was noteworthy that chloroplasts of the embryo bud had well developed giant grana under invisible light. Their developmental pathway in sacred lotus, however, was different from those of other higher plants grown under sunlight, intermittent light, or even in dark conditions (Fig. 1). The chloroplast development of embryo buds in Sacred lotus seeds in invisible light underwent only in the following three stages: (1) In the first stage the development was similar to that from other higher plants, the inner envelope membranes of the proplastids were invaginating. (2) In the second stage, a proplastid centre composed of prolamellar bodies (PLB)with semicrystalline structure was formed, and was accompanied by one or two huge starch grains in almost each proplastid. In the meantime, prothylakoid membranes extended parallelly from the plastid centre in three forms: (a) One plastid centre extending parallelly prothylakoid membranes from itself in one direction; (b) The same to (a), but extending in two directions; (c) Two plastid centres extending parallelly prothylakoid membranes between the centres. (3) In the third stage, grana and stroma thylakoid membranes of chloroplasts were formed. It is to be noted that most of chloroplasts had only one or two giant grana which often extended across the entire chloroplast body, and the length of the grana thylakoid membranes of the chloroplasts from embryo bud in Sacred lotus is 3 to 5 times as many as that in other higher plants. However, their stromatic thylakoid membranes were rather rare and very short. The giant grana were squeezed to the margin of the chloroplast envelope by one or two huge starch grains.     

DEVELOPMENT OF THE DIMORPHIC CHLOROPLASTS OF SUGAR CANE

The vascular bundle sheath cells of sugar cane contain starch-storing chloroplasts lacking grana, whereas the adjacent mesophyll cells contain chloroplasts which store very little starch and possess abundant grana. This study was undertaken to determine the ontogeny of these dimorphic chloroplasts. Proplastids in the two cell types in the meristematic region of light-grown leaves cannot be distinguished morphologically. Bundle sheath cell chloroplasts in tissue with 50% of its future chlorophyll possess grana consisting of 2-8 thylakoids/granum. Mesophyll cell chloroplasts of the same age have better developed grana and large, well structured prolamellar bodies. A few grana are still present in bundle sheath cell chloroplasts when the leaf tissue has 75% of its eventual chlorophyll, and prolamellar bodies are also found in mesophyll cell chloroplasts at this stage. The two cell layers in mature dark-grown leaves contain morphologically distinct etio-plasts. The response of these two plastids to light treatment also differs. Plastids in tissue treated with light for short periods exhibit protrusions resembling mitochondria. Plastids in bundle sheath cells of dark-grown leaves do not go through a grana-forming stage. It is concluded that the structure of the specialized chloroplasts in bundle sheath cells of sugar cane is a result of reduction, and that the development of chloroplast dimorphism is related in some way to leaf cell differentiation.     

PIGMENTS AND PLASTIDS IN CULTURES OF TOTIPOTENT CARROT CELLS

Cells from a strain of carrot which was prone to form deep-seated chlorophyll in its storage organ have been cultured in a manner that promoted them to organize into plantlets. Whereas the free cells contained only chloroplasts, the plantlets derived from these cells formed all types of plastids (“proplastids,” leucoplasts, chromoplasts, and chloroplasts) in accordance with the location of the cells in question in the developing plant body. The developmental history of the plastids has been traced with the electron microscope. The events of chloroplast development, previously described by Israel and Steward (1967) for cultured carrot explants, have been verified. The bearing of this new evidence upon the control of plastid development and biochemistry is discussed and related to other recent studies. The conclusion is that all totipotent carrot cells have plastids as essential organelles but that their final form and content are sharply defined by the factors inherent in the location of the cells in the plant body as it emerges.     

THE ULTRASTRUCTURAL DEVELOPMENT OF THE DIMORPHIC PLASTIDS OF ZEA MAYS L.

The development of the dimorphic chloroplasts of Zea mays L. in adult foliage leaves is described, and a method of correlating ultrastructural stages by means of leaf chlorophyll is presented. In addition, the developmental changes in chlorophyll a/b ratio are discussed. Both the mesophyll and the bundle sheath plastids contain small grana at the earliest stages of plastid development. As the plastids enlarge, the mesophyll grana stacks increase in both length of the appressed membrane and in the number of thylakoids per granum. Initially, the grana stacks in the bundle sheath plastids also enlarge, but as the plastids approach full size, most of the membrane appression is lost. However, the remaining areas of appression in the bundle sheath plastids show an increase in the number of thylakoids in each small granum.     

Studies on the Conversion of Chloroplast to Chromoplast During Fruit Ripening in Solanum pseudo-capsicum var. diflorum

Determination of chlorophyll and carotenoid contents in the ectocarp during fruit ripening in Solanum pseudo-capsicum var. diflorurn (Veil.) Bitter revealed that the changes of fruit colour coincided with the decline of chlorophyll and the increase of carotenoid contents. The conversion of chloroplasts to chromoplasts in the fruit was studied by electron microscopy. The early green fruit was characterized by chloroplasts with a typical grana-intergranal thylakoid structure. At yellow-green fruit stage the thylakoid system was disintegrated and replaced by few non-chlorophyllous single thylakoids, with accumulation of large osmiophilic plastoglobules. The plastids developed as the so-called proplastids. These indicated dedifferentiation of chloroplasts in a ripening fruit. When the fruit reached its yellow stage, numerous large plastoglobules contained in the young chromoplasts frequently showed transitional changes to plastid tubule structure. At first, the center of plastoglobules became semi-translucent. It was believed that the young chromoplast were in an initial state of carotenoid deposition, followed by plastoglobules elongation and tubule protrution from the globules. These tubules were surrounded with an electron dense membranous sheath leaving the core semi-translucent. Concurrently a series of vesicles in different developmental stages appeared from the stroma of the plastid, likely representing a process of formation of numerous small new plastoglobules. In the chromoplasts of a ripe orange-or orange red-colored fruit only numerous tubules and small plastoglobules were present. The plastid tubules increased in number and elongated in length filling the mature chromoplast. Numerous small plastoglobules also increased and distributed in the spaces between tubules. These results indicated that the reconstruction of a mature chromoplast from a dedifferentiated plastid was really a form of redifferentiation, and it might be concluded that the conversion of chloroplast to chromoplast in the fruit of S. pseudo-capsicum var. diflorum, in fact, was a processes of dedifferentiation and redifferentiation.     

ULTRASTRUCTURAL CHANGES OF CHLOROPLASTS IN ATTACHED AND DETACHED,AGING PRIMARY WHEAT LEAVES

Degradation of chloroplasts is shown in mesophyll cells of primary leaves of wheat. The sequence of ultrastructural changes in chloroplasts of naturally senescing leaves is compared with that of detached, aging leaves. In chloroplasts of naturally senescing leaves, the first indications of aging are the appearance of osmiophilic globuli and reorientation of the thylakoidal system. The membranes of the grana and intergrana lamellae then become distended and later dissociate into distinct vesicles. Concurrent with these membrane changes, osmiophilic globuli increase in size and number, and the stroma breaks down. Finally, the chloroplast envelope ruptures and plastid contents disperse throughout the cell's interior. In chloroplasts of mesophyll cells in detached, aging leaves, initial changes also include appearance of osmiophilic globuli, but later stages of chloroplast degradation are different. The chloroplast envelope ruptures before the lamellae break down. Swelling of grana and intergrana lamellae is not pronounced and, additionally, the thylakoidal system degenerates without forming vesicles or numerous osmiophilic globuli. These differences in the sequence of chloroplast degradation indicate that naturally senescing leaves rather than detached, aging leaves should be used in studies of chloroplast senescence.     

THE CUP PLASTID OF NICOTIANA RUSTICA

Weier , T. E., and C. R. Stocking . (U. California, Davis.) The cup plastid of Nicotiana rustica. Amer. Jour. Bot. 49(1): 24–32. Illus. 1962.—In situ and isolated chloroplasts of Nicotiana rustica have been studied by light and electron microscopy. Under certain conditions, notably of low light intensity, the starch-free plastid forms a cup. In isolated plastids the form may be modified by the tonicity of the isolation medium. In situ, the cup always faces the cell wall. Electron micrographs show the cup to be formed of compartmented grana connected at irregular intervals by flexuous channels known as frets. The interior of the cup is filled with a finely granular stroma which also forms the material surrounding the grana and the frets in the body of the cup. The grana radiate outward from the central stroma. They may be considered as cylinders. They are fairly rigid, as curvatures to form the cup-shape occur only in the interconnecting fretwork. The compartments may have a limited movement with reference to their axis. These evidences of movement of the part of the ultramicro plastid structures thought to contain chlorophyll suggest that the movement may be related to changes in light intensity or other factors influencing the rate of photosynthesis.     

CHLOROPLAST DEVELOPMENT IN THE GERMINATING SAFFLOWER (CARTHAMUS TINCTORIUS) COTYLEDON

Proplastids in the mesophyll cells of the cotyledons of mature seeds of safflower are irregular in shape and compressed in narrow corners between the large inclusion bodies, oil vacuoles and protein bodies. The proplastids contain a few irregular internal membranes. During dark germination, sheets or sac-like membranes are produced by the activity of the inner component of the proplastid envelope. These continuous membranes become reticulate and aggregate to the center of the proplastid to form after seven days' germination a quasicrystalline prolamellar body. The membranes are at first irregularly arranged and are of two sorts: those found in the interior of the developing prolamellar body, composed of laterally connected spherical profiles, and those on the periphery of the prolamellar body, which are continuous smooth sheets. The prolamellar body in these dark-germinated proplastids reverts after 3 hr of illumination to the irregularly arranged membranous structure of the 5-day dark germination stage. After 6 hr of illumination membranes grow from the prolamellar body forming concentric loops which, in cross section, appear as concentric circles. These membranes must be nested semi-spheroids. Small grana appear immediately on these looped membranes close to the prolamellar body. With further illumination additional grana develop along the looped membranes in close proximity to the slowly disappearing prolamellar body. Grana increase in size and number along the looped intergranal membranes. The prolamellar body disappears after 15 hr of illumination. The interconnecting fret membranes, sparse at the 15-hr stage, increase and after 24-hr illumination result in the typical grana fretwork system of the mature chloroplast. Membranes are continuously being produced by the invagination of the inner member of the plastid envelope.     

Structure and function of tomato leaf chloroplasts during ammonium toxicity

Ammonium toxicity resulted in morphological modifications of tomato leaf chloroplasts. The chloroplasts, which are normally flattened around the protoplast periphery, became ellipsoidally rounded and dispersed through the protoplasm. The first apparent effect of plastid degradation was development of many vesicles from the fretwork. Later the grana lamellae swelled, and some disappeared. Eventually, distinct grana could not be detected.

Ammonium accumulation, chlorophyll loss, and photosynthetic decrease occurred simultaneously. Initial changes in these processes preceded the detection of modifications of fine structure; however, each continued with further breakdown of the chloroplasts.

     

Effect of cytokinins on plastid development and photosynthetic polypeptides during organogenesis ofPinus ponderosa Dougl. cotyledons culturedin vitro

InPinus ponderosa Dougl., application of the cytokinins, benzyladenine and 2-isopentenyl adenine, to excised cotyledons, promoted thein vitro formation of meristematic centers which led to bud and shoot production. Meristematic cells showed plastids with poorly developed thylakoid membranes and rudimentary grana, whereas cells in non-meristematic tissues and in growth regulator free medium, had chloroplasts with well developed inner membranes, and more thylakoid membranes and grana than plastids of meristematic cells. Chlorophyll and six polypeptides associated with photosynthesis were present in lower concentrations in cytokinin-treated cotyledons than in those cultured in growth regulator free medium. Both benzyladenine and 2-isopentenyl adenine are effective in inhibiting the accumulation of at least two photosynthetic polypeptides in the first 24 h in culture. The ability of cotyledons to respond in this way to cytokinins is lost after three days in culture in growth regulator free medium prior to treatment with cytokinin.     

Rice NON-YELLOW COLORING1 is involved in light-harvesting complex II and grana degradation during leaf senescence

Chlorophyll degradation is an aspect of leaf senescence, which is an active process to salvage nutrients from old tissues. non-yellow coloring1 (nyc1) is a rice (Oryza sativa) stay-green mutant in which chlorophyll degradation during senescence is impaired. Pigment analysis revealed that degradation of not only chlorophylls but also light-harvesting complex II (LHCII)-bound carotenoids was repressed in nyc1, in which most LHCII isoforms were selectively retained during senescence. Ultrastructural analysis of nyc1 chloroplasts revealed that large and thick grana were present even in the late stage of senescence, suggesting that degradation of LHCII is required for the proper degeneration of thylakoid membranes. Map-based cloning of NYC1 revealed that it encodes a chloroplast-localized short-chain dehydrogenase/reductase (SDR) with three transmembrane domains. The predicted structure of the NYC1 protein and the phenotype of the nyc1 mutant suggest the possibility that NYC1 is a chlorophyll b reductase. Although we were unable to detect the chlorophyll b reductase activity of NYC1, NOL (for NYC1-like), a protein closely related to NYC1 in rice, showed chlorophyll b reductase activity in vitro. We suggest that NYC1 and NOL encode chlorophyll b reductases with divergent functions. Our data collectively suggest that the identified SDR protein NYC1 plays essential roles in the regulation of LHCII and thylakoid membrane degradation during senescence.     

Ultrastructure and Isozyme Analysis of Cultured Soybean-Nicotiana Fusion Products

Protoplasts were prepared from 2 days old subcultures of soybean (Glycine max (L.) Merr.) and fragments of young leaves of tobacco (Nicotiana tabacum var. “Xanthi”) according to the methods of Kao. Protoplasts were fused and single fusion products were cultured in Cuprak dishes as previously described. Fusion products were fixed and embedded in plastic by reported methods for electron microscope study. Isoenzyme studies were carried out according to described methods. Proteins were electrophoresed on 5% polyacrylamide gels and stained. Fusion products were easily identified on the basis of the presence of both tobacco chloroplasts and soybean leucoplasts (Fig. 1). The chloroplasts contained typical grana and stroma lamellae; leucoplasts were characterized by numerous starch granules and a paucity of internal lamellae. After 15 hours in culture, thorough mixing of cytoplasm had occurred as evidenced by the distribution of plastids. Fusion of interphase nuclei was not observed in any of the fusion products. Premitotic nuclear fusions which have been reported previously may signify unhealthy fusion products. Fusion products underwent their first cell division within 2–3 days in culture; divi- ding nuclei contained complete sets of both tobacco and soybean chromosomes. During subsequent divisions, hybrids gradually lost some tobacco chromosomes. By 4.5 days, small clusters of hybrid cells were evident. The chloroplasts of such hybrid cells exhibited unusual shapes, possibly as a result of starch accumulation (Fig. 2b, 2c). The leucoplasts remained unchanged. Within 2 weeks, hybrid clusters contained 100–200 cells. Very few chloroplasts were detected in these cells by electron microscopy. The chloroplasts present were highly modified. Typically, these plastids were characterized by enlarged grana and elongated parallel stacks of stroma lamellae. Similar changes in plastid morphology were observed in pea-soybean fusion products cultured for 1 week. It is not possible to determine from the present study whether chloroplasts were being diluted during cell proliferation or whether they were dedifferentiating. Previous ultrastructural research suggests that dedifferentiation of chloroplasts occurs in fusions involving similar species while chloroplasts degeneration is more likely in fusions of widely separated species . Biochemical evidence from studies of the electrophoretic mobility of the plastid-encoded large subunit of ribulose-1, 5-bisphosphate carboxylase and the endonuclease restriction patterns of plastid DNA indicate that plastids may either assort randomly or both plastid types may coexist in cells of regenerated hybrid plants. Chloroplasts were not detected in hybrids cultured for prolonged periods. The leucoplasts in these cells were indistinguishable from leucoplasts of parental soybean cells. Leucoplasts were not diluted during cell division and their numbers were likely maintained by plastid division. Over 20 hybrid cell lines were established and cultured for 7–9 months. Chromosome analysis revealed that many lines including the one illustrated in Fig. 4 retained over one half of the tobacco chromosomes in addition to the full soybean chromosome complement . Zymograms from this same cell line are presented in Fig. 5. The electrophoretic patterns for both dehydrogenases clearly demonstrate that hybridization has been achieved. The shikimate dehydrogenase (SDH) zymogram for the hybrid shows that the broad slow- moving band from soybean and the 2 distinctive fast-moving bands found in Nicotiana are all present in the hybrid. Similarly, for 6-phosphogluconate dehydrogenase (6-PGDH), the hybrid contained the bands from soybean and the 3 slower-moving bands from Nicotiana as well as one of the 2 fast-moving bands found in the latter. This study demonstrates the usefulness of both electron microscopy and isozyme analysis for examining hybrid cells derived from plant protoplast fusion. During the early stages of hybrid culture when small sample size precludes isozyme analysis, ultrastructure studies permit the identification of hybrid cells, after prolonged culture, the isozyme technique is a much more sensitive measure of hybridization than is electron microscopy.     

EFFECTS OF DARKNESS AND OF STREPTOMYCIN ON THE FINE STRUCTURE OF EUGLENA GRACILIS

Siegesmund , Kenneth A., Walter G. Rosen , and Stanley R. Gawlik . (Marquette (J., Milwaukee, Wis.) Effects of darkness and of streptomycin on the fine structure of Euglena gracilis. Amer. Jour. Bot. 49 (2) : 137–145. Illus. 1962.—Dark-grown Euglena gracilis cells, transferred from streptomycin (SM)-containing medium to SM-free medium 5 days before transfer to light, turn green normally, indicating that proplastids are unaffected by SM. SM-bleached cells, grown in light, contain numerous bodies composed of concentric lamellae (CL bodies). These differ from chloroplasts in that their lamellae lack electron-dense dots, are not coalesced, and they lack a 3-layered limiting membrane and pyrenoids. CL bodies are absent from dark-grown normal and dark-grown SM-bleached cells, as well as from light-grown normal cells. It is suggested that CL bodies result from a derangement of chloroplast synthesis caused by SM blockage of chlorophyll synthesis.     

THE GREENING OF ETIOLATED BEAN LEAVES AND THE DEVELOPMENT OF CHLOROPLAST FINE STRUCTURE IN ABSENCE OF PHOTOSYNTHESIS

CMU inhibits oxygen evolution in greening etiolated bean leaves.In the presence of this compound chlorophyll content is reducedand fine structure development of the chloroplasts is markedlyaffected. The number of grana per chloroplast is reduced butthe grana are larger and contain more thylakoids than the granain chloroplasts of the greening control leaves. Sucrose reversesthe effect of CMU on pigment content and fine structure developmentof chloroplasts. (Received September 14, 1965; )