Developmental Aspects of Ultrastructure and Histochemistry of the Stylar Transmitting Tissue of Nicotiana sylvestris

The structure and histochemistry of the solid style of Nicotiana sylvestris Speg. and Comes have been studied by light and electron microscopy. The transmitting tissue develops large intercellular spaces filled with secretions rich in proteins and carbohydrates during maturation. The cells possess large nuclei, numerous plastids with starch grains, mitochondria, ribosomes and well developed endoplasmic reticulum and golgi apparatus. The plastids in the stylar region immediately below the stigma produce electron-dense osmiophilic substances which are probably transferred into the cytoplasm by a process resembling budding-off of vesicles. The Golgi apparatus may use the starch grains as a source of sugars for the synthesis and secretion of extracellular polysaccharides. The structural and cytochemical differences between the glandular cells of the stigma and the stylar transmitting tissue are discussed.     

野生烟草花粉活力与柱头可授性及繁育特性研究

采用TTC法测定2个野生烟草材料(花烟草、哥西氏烟草)和1个栽培品种(K326)的花粉活力及其日变化情况,通过联苯胺-过氧化氢法测定3个烟草材料的柱头可授性、利用直接授粉法测定不同开花天数柱头可授性变化,并通过估算花粉胚珠比(P/O)、杂交指数(OCI)及授粉试验分析3个烟草材料的繁育特性。结果表明:(1)哥西氏烟草的花粉活性(74.9%)显著高于K326(52.2%)和花烟草(45.3%),且K326与花烟草间差异不显著;3个烟草材料花粉活力日变化均呈双峰曲线,峰值分别在13:00与15:00左右,且3个烟草材料的花粉活力日最低值与气温日最高值同时出现在14:00。(2)K326柱头可授性显著高于花烟草和哥西氏烟草,且2个野生烟草间可授性无显著差异;不同烟草的最佳授粉时期不同,哥西氏烟草自开花前1d至花后4d,一直保持较高的柱头可授性;花烟草的最佳授粉时期为花后2~3d;K326的柱头在开花前1d至开花后1d授粉最佳。(3)K326以自交为主,存在异交现象;哥西氏烟草繁育类型为兼性异交,自交亲和;花烟草繁育以异交为主,自交亲和性差。研究认为,野生烟草柱头可授性显著低于栽培品种从而影响其结实性,花烟草结实率低主要是由自交不亲和性造成的,而缺乏有效的传粉机制是造成哥西氏烟草结实性差的主要原因。     

烟台甜樱桃柱头的可授性、形态特征与坐果率

采用数码照相、电镜扫描、联苯胺-过氧化氢测试及去雄套袋等技术手段, 对烟台甜樱桃(Cerasus avium)花期不同发育阶段柱头的可授性、形态特征和坐果状况进行了观察。结果表明, 烟台甜樱桃在套袋状态下, 柱头可授期从开花前1天开始可持续5–7天。从杯状花期到花瓣平展期, 柱头逐渐有乳突细胞破裂并呈现分泌液, 出现渐强的可授性; 从花瓣平展期到花瓣脱落期, 柱头由暗黄渐变至暗黑, 逐渐萎缩并丧失可授性。去雄套袋及人工授粉实验结果显示, 在大蕾期、杯状花期、花瓣展放期、花瓣平展期和花瓣脱落期进行人工授粉, 烟台甜樱桃的坐果率分别为60.50%、58.33%、62.08%、57.14%和39.13%。在自然条件下烟台甜樱桃的坐果率一般为30%–42%, 传粉成功的最佳期主要发生在杯状花期至花瓣平展期。     

烟台甜樱桃柱头的可授性、形态特征与坐果率

采用数码照相、电镜扫描、联苯胺-过氧化氢测试及去雄套袋等技术手段,对烟台甜樱桃（Cerasus avium）花期不同发育阶段柱头的可授性、形态特征和坐果状况进行了观察。结果表明,烟台甜樱桃在套袋状态下,柱头可授期从开花前1天开始可持续5–7天。从杯状花期到花瓣平展期,柱头逐渐有乳突细胞破裂并呈现分泌液,出现渐强的可授性;从花瓣平展期到花瓣脱落期,柱头由暗黄渐变至暗黑,逐渐萎缩并丧失可授性。去雄套袋及人工授粉实验结果显示,在大蕾期、杯状花期、花瓣展放期、花瓣平展期和花瓣脱落期进行人工授粉,烟台甜樱桃的坐果率分别为60.50%、58.33%、62.08%、57.14%和39.13%。在自然条件下烟台甜樱桃的坐果率一般为30%–42%,传粉成功的最佳期主要发生在杯状花期至花瓣平展期。     

The chloroplast genome of Nicotiana sylvestris and Nicotiana tomentosiformis: complete sequencing confirms that the Nicotiana sylvestris progenitor is the maternal genome donor of Nicotiana tabacum

The tobacco cultivar Nicotiana tabacum is a natural amphidiploid that is thought to be derived from ancestors of Nicotiana sylvestris and Nicotiana tomentosiformis. To compare these chloroplast genomes, DNA was prepared from isolated chloroplasts from green leaves of N. sylvestris and N. tomentosiformis, and subjected to whole-genome shotgun sequencing. The N. sylvestris chloroplast genome comprises of 155,941 bp and shows identical gene organization with that of N. tabacum, except one ORF. Detailed comparison revealed only seven different sites between N. tabacum and N. sylvestris; three in introns, two in spacer regions and two in coding regions. The chloroplast DNA of N. tomentosiformis is 155,745 bp long and possesses also identical gene organization with that of N. tabacum, except four ORFs and one pseudogene. However, 1,194 sites differ between these two species. Compared with N. tabacum, the nucleotide substitution in the inverted repeat was much lower than that in the single-copy region. The present work confirms that the chloroplast genome from N. tabacum was derived from an ancestor of N. sylvestris, and suggests that the rate of nucleotide substitution of the chloroplast genomes from N. tabacum and N. sylvestris is very low. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

白杨派树种雌蕊柱头可授性及其检测方法的研究

以银腺杨、毛新杨和银毛杨为材料,采用定期授粉观察以及联苯胺-过氧化氢染色等方法,对白杨派树种雌蕊柱头可授性进行了研究,结果表明:在温室水培条件下,白杨树种雌蕊柱头的可授期一般在1~3 d内,其中毛新杨雌蕊柱头可授期比银腺杨和银毛杨相对短些.但3个树种进入最佳授粉时期时的柱头开裂角度约为180°,此时柱头发亮且表面有大量分泌物存在;在适宜授粉时期内,用联苯胺-过氧化氢染色后柱头变蓝,并有气泡产生。在育种实践中,可根据柱头形态以及联苯胺-过氧化氢染色后柱头颜色变化和气泡产生情况及时判别有关白杨派树种最佳授粉时期。     

Plasmatubules in the pollen tubes of Nicotiana sylvestris

Ultrastructural studies of the pollen tubes of Nicotiana sylvestris grown in the pistil revealed an extensive development of plasmatubules formed by evaginations of the plasma membrane. The plasmatubules occurred as twisted tubular structures in the periplasmic space along the tube wall and, in cross section, exhibited circular profiles with an outer diameter of 28±4 nm. They were also seen in deep, pocket-like invaginations of the plasma membrane and in this case the profiles had an outer diameter of 34±8 nm. In the pocket-like invaginations they were partially branched and often closely packed to form groups with obvious patterns. The enlargement of the plasma-membrane area resulting from plasmatubules formed along the tube wall was about six-to tenfold. Pollen tubes grown in vitro exhibited poorly developed plasmatubules. It is suggested that the large extension of the plasma membrane could enhance the uptake of nutrients, and thus might be responsible for the comparatively fast growth of pollen tubes in the pistil. Moreover, it is also assumed that the turnover rate of the Golgi apparatus must be higher in pollen tubes growing in vivo than in vitro, in order to provide a sufficient amount of membrane for the formation of the plasma membrane with its tubular modifications.     

The Anatomy, Histochemistry and Ultrastructure of Stigmas and Styles in Commelinaceae

The anatomy and ultrastructure of stigmas in 37 species of 13genera of Commelinaceae are described. The stigmas are papillate,papillae forming a dense fringe of cells around the mouth ofthe stylar canal in most species. The papillar cell wall iscovered by an unstructured cuticle of variable thickness andis of variable thickness because of small wall ingrowths. Thecuticle and the external surface of the papillar cell wall arevariably disrupted, particularly in the mid and basal regionsof the cell. This was not found in species of the genus Aploleiaor Callisia. The cell cytoplasm possesses all major organellesexcept chloroplasts and each cell is vacuolate. In all species except Aploleia mulitiflora the style comprisesan epidermis, a cortex and a hollow, tripartite canal whichis continuous into the ovary cavity. The three vascular strandsare positioned at the apex of each canal lobe. The canal cellsare elongate and tabular and the wall abutting the canal hasingrowths. The style in Aploleia is solid and the transmittingtissue comprises cells whose walls are electron opaque. Thecytoplasms of both types of cell are similar in content althoughthere is a single, large vacuole in canal cells and many smallvacuoles in transmitting tissue. The morphology, position and histochemistry of stigmatic andstylar exudate was similar in all ‘wet’ stigmas.Most of the exudate originates from the stylar canal althoughsignificant contributions are made by the papillae in stigmasof Coleotrype, Dichorisandra and Thyrsanthemum. There is no apparent relationship between stigma structure andthe presence of self-incompatibility. Stigma papillae, stylar canal, transmitting tissue, Commelinaceae     

Amplification of repetitive DNA from Nicotiana plumbaginifolia in asymmetric somatic hybrids between Nicotiana sylvestris and Nicotiana plumbaginifolia

Summary Asymmetric somatic hybrids were obtained between a chlorophyll-deficient mutant of Nicotiana sylvestris (V42) and a nitrate-reductase (NR)-deficient line of N. plumbaginifolia (cnx20 or Nia26), using each of the parents alternately as the irradiated donor. Irradiation doses applied ranged from 10 to 1,000 Gy of gamma-rays. Hybrid selection was based on complementation of NR deficiency with wild-type NR genes. To aid in the analysis of somatic hybrids, species-specific repetitive DNA sequences from N. plumbaginifolia (NPR9 and NPR18) were cloned. NPR18 is a dispersed repetitive sequence occupying about 0.4% of the N. plumbaginifolia genome. In turn, NPR9, which is part of a highly repetitive DNA sequence, occupies approximately 3% of the genome. The species-specific plant DNA repeats, together with cytological analysis data, were used to assess the relative amount of the N. plumbaginifolia genome in the somatic hybrids. In fusion experiments using irradiated N. plumbaginifolia, an increase in irradiation dose prior to fusion led to a decrease in N. plumbaginifolia nuclear DNA content per hybrid genome. For some hybrid lines, an increase in the quantity of repetitive sequences was detected. Thus, hybrid lines 1NV/21, 100NV/7, 100NV/ 9, and 100NV/10 (where N. plumbaginifolia was the irradiated donor) were characterized by amplification of NPR9. In the reverse combination (where N. sylvestris was the irradiated donor), an increase in the copy number of NPR18 was determined for hybrid clones 1VC/2, 1VC/3, 100VC/2 and oct100/7. Possible reasons for the amplification of the repeated sequences are discussed.     

花发育阶段以及温度对不同品种甜樱桃柱头可授性的影响

以‘布鲁克斯’、‘美早’、‘红蜜’等5个甜樱桃(Prunus avium)品种为试材,检测不同品种甜樱桃花期不同发育阶段的柱头可授性;并利用人工气候箱模拟花期低温(2 ℃/10 ℃)、常温(6 ℃/18 ℃)、高温(12 ℃/24 ℃)以及超高温(12 ℃/24 ℃/30 ℃)环境,研究不同温度处理对甜樱桃柱头可授性的影响。结果表明：(1)‘美早’、‘先锋’和‘萨米脱’适宜的授粉温度是常温(6 ℃/18 ℃)环境,‘红蜜’适宜的授粉温度是高温(12 ℃/24 ℃)环境,‘布鲁克斯’适宜授粉的温度范围更广,在常温(6 ℃/18 ℃)和高温(12 ℃/24 ℃)环境条件下其柱头均具有较高的活性。(2)‘红蜜’柱头对花期低温(2 ℃/10 ℃)和超高温(12 ℃/24 ℃/30 ℃)的抗性较强;‘布鲁克斯’和‘萨米脱’柱头对花期超高温(12 ℃/24 ℃/30 ℃)抗性较差;‘美早’柱头对花期低温(6 ℃/10 ℃)的抗性较强,但对花期高温(12 ℃/24 ℃)的抗性较差;‘先锋’柱头对花期低温(6 ℃/10 ℃)和超高温(12 ℃/24 ℃/30 ℃)均无明显抗性。研究认为,低温(2 ℃/10 ℃)对甜樱桃柱头可授性的影响主要在开花之后,而超高温(12 ℃/24 ℃/30 ℃)对整个开花过程柱头可授性的影响都很大。     

膏桐花粉活力与柱头可授性及其生殖特性研究

采用TTC法测定了膏桐花粉的活力和寿命,用联苯胺-过氧化氢法测定其柱头可授期,以套袋结果检测花粉活力与柱头可授性的变化,以研究柱头颜色变化与柱头可授性的关系以及开花式样对生殖成功的影响.结果表明:(1)膏桐雄花单花花期一般为2 d,花后0~9 h内花粉活力相对较高,24~33 h后花粉活力较低,48 h后花粉基本无活力.(2)膏桐雌花单花期为5~12 d,柱头可授性在花后1~4 d最强,5~8 d可授性开始逐渐降低,第9天基本失去可授性.(3)膏桐柱头表面无明显的渗出物,柱头的可授性与柱头颜色的变化相关,柱头绿色越多,柱头可授性越强;遇降雨时,少量雌花的花瓣展开而柱头仍呈球状,或者花瓣不展开而柱头先伸出,此时柱头可授性较差.(4)膏桐常表现为雄性先熟,尽管雌花数目较少,但开放速率较快,且集中在第3~5天开放,雌花开放完毕也仍有雄花未开放,为展开后的柱头留有较长时间等候昆虫传送花粉,为保障生殖成功提供了机会,但少数花序先开雌花,后开雄花,有支持异交而降低自交的倾向.     

Development, Histochemistry and Ultrastructure of Gum-resin Ducts in Commiphora mukul Engl.

Gum-resin ducts are present in the primary and secondary phloemof Commiphora mukul Engl. The important gum-resin, known commerciallyas ‘guggul’, is secreted and collected in ductswhich develop schizogenously. The duct initials have dense cytoplasm,large nuclei, increased cytoplasmic RNA and proteins. The lumenof newly-formed ducts widens accompanied by anticlinal divisionsand subsequent tangential elongation of epithelial cells. Histochemicaltests reveal that the epithelial cells have apparently largeamounts of proteins, cytoplasmic RNA, and DNA in the nucleus.Lipid globules are also present in these cells. Epithelial cellwalls in contact with the duct are thin and of a loose fibrillarmesh. Microtubules, randomly oriented in the epithelial cellsare always parallel and adjacent to the wall. The cytoplasmis rich in ribosomes, endoplasmic reticulum, mitochondria, plastidsand vacuoles containing osmiophilic substances. At the peripheralregion of the duct, electron-transparent bodies containing densely-stainedmaterial are present close to the tangential wall.     

Two Feedback-Insensitive Enzymes of the Aspartate Pathway in Nicotiana sylvestris

Lysine and threonine overproducer mutants in Nicotiana sylvestris, characterized by an altered regulation of, respectively, dihydrodipicolinate synthase and aspartate kinase activities, were crossed to assess the effects of the simultaneous presence of these genes on the biosynthesis of aspartate-derived amino acids. The monogenic dominant behavior of both resistance traits was confirmed, and their loci were found to be unlinked. Study of the inhibition properties of dihydrodipicolinate synthase and aspartate kinase activities in RAEC-1 × RLT 70 confirmed the heterozygote state of both mutations, because only half of their lysine-sensitive activity could still be inhibited by this negative effector. Analysis of the free amino acid pool during the growth of the double mutant revealed a major free lysine overproduction reaching up to 50% of the total pool, whereas the other aspartate-derived amino acids remained equally or even less abundant than in the wild type. An abnormal phenotype was clearly associated with such high levels of lysine accumulation, which points out the possible role of this amino acid in the developmental features of the plant. Comparison of the amino acid content, free and total (free + protein-bound), between the wild type, the two mutants, and the double mutant obtained by crossing them brings new insights on the regulation of the aspartate pathway, and on its implications in relationship to plant nutritional value improvement.     

Somatic hybridization between Nicotiana rustica and N. sylvestris

Protoplasts isolated from cell cultures of chlorophyll-deficient Nicotiana rustica cv. chlorotica and wild-type N. sylvestris were fused. The scheme for selection of somatic hybrids was based on the inability of the protoplast-derived colonies of the parental species to turn green; N. sylvestris protoplasts also had a very low plating efficiency in the medium employed. A total of 777 green colonies which were presumptive hybrids were isolated within four weeks of the fusion experiments. One hundred and eight green colonies formed shoots in vitro and 16 lines were rooted and grown in the greenhouse. Each of these hybrid plants displayed vegetative and floral traits intermediate to those of the parental species, except for plant height which in almost all cases was greater in the hybrids. Isozyme analyses by gel electrophoresis and isoelectric focussing of the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (RUBPCase) demonstrated that the nuclear genomes of both parents were expressed by the hybrids. Each of the eight somatic hybrid plants analyzed expressed only the N. rustica chloroplast genome as shown by isoelectric focussing of the large subunit of RUBPCase. This study demonstrated the value of N. rustica cv. chlorotica as a parental line in somatic hybridization with N. sylvestris and it might have widespread use with wild-type lines of other species.     

Isolation of antibiotic resistant variants in a higher plant,Nicotiana sylvestris

Using cell suspension cultures of Nicotiana sylvestris, spontaneous oligomycin- or chloramphenicol-resistant variants were isolated at a frequency of 10⁻⁷. Treatments with ethyl methane sulfonate (EMS), a mutagen, increased this frequency 5–50 times. After 18–24 months of culture on basal medium, 6 variants retained a low level of resistance to oligomycin, 9 a high level. Four chloramphenicol-resistant variants were still slightly resistant and one was three times more resistant than the wild-type.     

Developmental and Evolutionary Aspects of the Neurohypophysis

Although phylogenetic studies of neurohypophysial principlesare well advanced, studies of their development are rare. Fetalmammals have been shown to have unusually high vasopressor/oxytocicratios in their pituitaries. Paper chromatography of extractsfrom pituitaries of fetal sheep and fetal seals (Callorhinusursinus) indicated the presence of AVT, in addition to AVP andoxytocin. This was confirmed by the pharmacological and chemicalanalysis of AVT, purified from fetal seal pituitaries. The presenceof AVT suggested a "recapitulation" of a principle previouslyknown only in sub-mammalian vertebrates. Studies of ammocoetelarvae and adult lampreys (Lampetra nchardsoni) showed the presenceof AVT, but there was no evidence for any "recapitulation" ofan "early" neutral principle. It seemed possible that an apparentrecapitulation might occur only when the principle had a usein fetal life. Therefore the possible actions of AVT (and AVP)in the mammalian fetus (guinea pig) were studied. AVT and AVPwere shown to act on the isolated amnion to slow, and even reversefetal-maternal water movement. AVP acted on the yolk sac, thefetal skin and the fetal bladder to increase water flow inwards,towards the fetus. These actions suggested an extraplacentalmechanism for supplying water to the fetus and its environment,through the extraembryonic membranes. These actions were reminiscentof those known in the anurans. The apparent success of the conceptof recapitulation, in both the molecular and functional fields,may have been fortuitous. However, the results suggest thatphylogenetic considerations may well be useful in understandingthe physiology of the mammal during fetal life.     

Ethylene and the regulation of senescence processes in transgenic Nicotiana sylvestris plants

BACKGROUND AND AIMS: Exposure of plants to ethylene can influence a spectrum of developmental processes including organ senescence and abscission. The aim of this study was to examine the role of the gaseous regulator in Nicotiana sylvestris plants exhibiting a silenced or constitutive ethylene response. METHODS: Transgenic N. sylvestris plants were generated that either ectopically expressed the Arabidopsis mutant ethylene receptor ETR1-1 or the tomato EIN3-like (LeEIL1) gene. Highly expressing homozygous lines were selected and the time-course of development, from germination to organ senescence, was studied. KEY RESULTS: Fifty percent of the homozygous Pro(35S):ETR1-1 lines examined showed a high susceptibility to collapse prior to flowering, with plant death occurring within a few days of leaf wilting. The time-course of leaf senescence in the remaining Pro(35S):ETR1-1 lines was visibly arrested compared to wild type (negative segregant) plants and this observation was reaffirmed by chlorophyll and protein analysis. Petal necrosis was also delayed in Pro(35S):ETR1-1 lines and corolla abscission did not take place. When senescence of Pro(35S):ETR1-1 plants did take place this was accompanied by leaf bleaching, but tissues remained fully turgid and showed no signs of collapse. A single Pro(35S):LeEIL1 line was found to exhibit consistently accelerated leaf and flower senescence and precocious flower bud shedding. CONCLUSIONS: These observations support a role for ethylene in regulating a spectrum of developmental events associated with organ senescence and tissue necrosis. Furthermore, the transgenic lines generated during this study may provide a valuable resource for exploring how senescence processes are regulated in plants.     

Induction of embryos in pollen cultures of Nicotiana sylvestris

Induction of embryos in pollen cultures was possible when young buds from plants induced to flower at 15°C were given a cold treatment at 10°C for 15 days. Following selective isolation of embryogenic pollen the cultures raised were given cold treatment at 10°C for 12 or more days. In such cultures the embryos appeared at a frequency of 1.2% of the cultured pollen on mineral-sucrose medium at pH 6.8.