Pollen Ultrastructure in Anther Cultures of Nicotiana tabacum: II. CHANGES ASSOCIATED WITH EMBRYOGENESIS

In ultrathin sections of 7- and 8-d-old anther cultures of Nicotianatabacum examined in the electron microscope, embryogenic pollenwas distinguished by the presence of zones in the cytoplasmof the vegetative cell consisting of multivesiculate bodiesresembling lysosomes. These zones increased with time, and in12-d-old samples (in which divisions of the vegetative cellwere about to commence), were almost devoid of contents. Ribosomesappeared to be virtually eliminated from the cell and many otherorganelles degraded; the few that remained, mainly plastids,were clustered around and in close contact with the vegetativenucleus. Plastid contents also declined with time, but mitochondrialstructure changed little. It is concluded that the gametophyticcytoplasm is destroyed before the first sporophytic divisionsof the vegetative cell. In the same sections, other structurally viable pollen whichlacked lysigenous zones was characterized by dense vegetativecytoplasm containing numerous small vacuoles. Mitochondrialand plastid structure differed from that of the embryogenicgrains, and in some instances lacked clear structural definition.Extensive stacks of rough e.r., which traversed the cytoplasmof the vegetative cell, were interpreted as assembly sites forexcess gametophytic protein and RNA synthesised during the cultureperiod.     

Pollen Ultrastructure in Anther Cultures of Nicotiana tabacum: III . THE FIRST SP.OROPHYTIC DIVISION

Ultrastructure of embryogenic tobacco pollen was examined atthe first division of the vegetative cell. Structurally emptyzones formed before division by the degradation of gametophyticcytoplasm were rapidly re-populated with ribosomes, mitochondria,dictybsomes, and organelles interpreted as lipid centres. Thelatter appeared to be active in synthesis, and were associatedwith a vesicular component. Starch accumulation also began atthe first division in plastids carried over from the mothercell. Each daughter cell possessed a typical primary wall confluentwith the inner layers of the intine. Plasmodesmata traversedthe wall between the two cells. The cells were either equalor unequal; equal divisions occurred in both the longitudinaland transverse planes. In many proembryoids, the generativecell showed signs of degeneration. The cell was either attachedto the intine or detached from it. Detached cells sometimesdivided. The data, are discussed in relation to other embryogenicand morphogenic systems induced from normally quiescent or highlydifferentiated cells.     

Embryoid Formation in Pollen Grains of Nicotiana tabacum

Anthers of Nicotiana tabacum (n = 24) were cultured on nutrientagar and examined at intervals for pollen embryoids. Embryoidswere formed in anthers of varying developmental stage, the youngestof which coincided with the liberation of free microspores fromtetrads, and the oldest with the formation of bicellular grains.This period in the development of the anther occupied 4–5days. Older anthers within this range were more successful thanyounger anthers. The first mitosis of the pollen was typicallyasymmetric and resulted in the formation of unequal generativeand vegetative cells. Some of the grains then went into a lagphase for at least 5–6 days, after which the mitotic conditionwas restored. Embryoids were formed by repeated division ofthe vegetative cell. If the generative cell divided, it didso only once or twice. Occasionally the first mitosis was symmetricand gave rise to equal cells, and in these instances both cellsprobably participated in embryoid formation. The youngest anthersexamined were probably less successful because fewer grainssurvived to enter mitosis. The number of embryoids produced varied considerably from oneanther to another both within the same bud and between differentbuds: values ranging from less than 400 to 10 000 per antherwere encountered. Most of these degenerated after the firstfew divisions, partly because they burst prematurely from thepollen grain wall. Embryoids which continued to develop formedplantlets and/or callus. The largest number of plantlets obtainedfrom one anther was 32. Haploid plantlets were also regeneratedfrom callus by transferring it to a low-sugar medium withoutauxin. The behaviour of grains not forming embryoids was also noted.     

Problems Encountered in the Culture of Isolated Pollen of a Burley Cultivar of Nicotiana tabacum

Pollen isolated from anthers of a Burley cultivar of N. tabacumby an anther homogenization procedure failed in culture to retainviability and produce plantlets even when isolation was precededby 14 d of anther preculture. By surgical manipulation of anthersprior to their culture it was shown that pollen viability wasvery sensitive to injury caused to the anther wall. Plantletscould be obtained however by culture of anthers in which oneor both pollen sacs had been carefully opened along the dehisenceline (DL). The somewhat lower yield of plantlets from such antherscompared to that from intact anthers is considered to resultnot from any change in the atmospheric environment of the pollenas a result of opening the pollen sac but from the inabilityin every case to keep the incisions strictly along the dehiscenceline. Pollen isolated from 14 d precultured anthers by this DL techniquewas able to yield plantlets when cultured in a simple definedmedium. However DL-isolated grains from 4 d precultured anthersfailed to retain viability in culture. It is concluded that pollen of this cultivar suffers severeinjury when isolated by the homogenization method and that thiscan be overcome by the DL technique. However conditions of culturehave not been established which reproduce for this cultivarthe nutritional conditions operating within the anther duringits early period in culture and which are essential to plantletformation.     

The Growth Activity and Fertilizing Capacity of Submersely Cultivated Pollen Tubes of Nicotiana tabacum L.

Tobacco pollen was cultivated in shaken suspension culturesand the growth estimated by weighing the mass of germinatedpollen separated from the nutrient solution. A formula for calculatingmean pollen tube length from the weight of the culture has beenderived for this pollen species. The growth of pollen tubes in vitro is shown to have a rhythmiccharacter. The rapid growth comparable to the mean growth ratein styles is limited to short time intervals alternating withprogressively extending periods of very depressed growth, whichceased entirely after 10–12 h of cultivation. When tested by placental pollination in vitro, the fertilizingcapacity of the pollen culture was found to increase duringthe first hour of cultivation but to decrease steadily thereafter.On the other hand, with the application of pollen tubes fromculture on stigma, a short precultivation period and even themere wetting of pollen had a negative effect on the seed set.With pollen cultivated longer than 4 h, no seed formation wasobserved.     

Pollen Dimorphism--Origin and Significance in Pollen Plant Formation by Anther Culture

Pollen dimorphism during the ripening of Nicotiana tabacum antherstakes the form of differentiation at the binucleate pollen stageinto normal (N) grains, characterized by their high frequency,larger size, densely–staining cytoplasm and high starchcontent and into smaller (S) grains characterized by their variableand low frequency and weakly–staining cytoplasm. Mostof the S grains show distinctive vegetative and generative nuclei(A grains); a small number have two vegetative–type nuclei(B grains). Evidence is presented that when excised anthersare cultured, pollen plants arise only from S grains. It issuggested that the differentiation into N and S grains arisesby an abnormal second meiotic division in the pollen mothercells. Nicotiana tabacum, tobacco, pollen dimorphism, anther culture     

Embryogenesis and Plantlet Formation through Direct Culture of Isolated Pollen of Nicotiana tabacum cv. Samsum and Nicotiana rustica cv. Rustica

Pollen embryos and plantlets of Nicotiana tabacum cv. Samsunand Nicotiana rustica cv. Rustica were obtained through directpollen culture without prior treatment or prior culture of anthersor buds. Isolated pollen was cultured first in a medium withoutsucrose, then transferred into Nitsch's H medium containing2% sucrose and 5 mM glutamine. The optimum medium for the initialculture was water and the optimum period of culture was ca.6 days when binucleate pollen was used. ¹ Present address: Friedrich Miescher Inst., P.O.B. 273, CH-4002Basel, Switzerland. (Received January 18, 1982; Accepted March 19, 1982)     

2,4-D-Sustained Photomixotrophic Growth of a Chlorophyllous Cell Suspension Culture of Nicotiana tabacum

A chlorophyllous, photomixotrophic cell suspension culture oftobacco (Nicotiana tabacum L.) was established using mediumcontaining 30 g/liter of sucrose and 1.5 µM 2,4-D. The2,4-D-sustained photomixotrophic line was able to show rapidregreening in the light after bleaching in the dark and characterizedwith a much slower and longer growth cycle than a heterotrophicline derived from the same original callus (cell doubling timeof 100 h vs. 40 h and duration of logarithmic phase of 17 daysvs. 7 days). The photomixotrophic line took up sucrose morerapidly than the heterotrophic line and accumulated starch duringthe early logarithmic phase when it showed a maximum photosyntheticcapacity on a chlorophyll basis (6.3µmol O₂/min/mg Chl).Chlorophyll content and photosynthetic capacity on a per cellbasis and on a cell fresh weight basis, on the other hand, decreasedduring this phase and reincreased later to reach maximum levels(310 µg Chl/g fr wt; 1.4 µmol O₂/min/g fr wt) whenthe line exhibited the highest activities of dark respiration(1.0 µmol; O₂/min/g fr wt) and cell division (mitoticindex of 3.0%). These characteristics of the photomixotrophicline were lost if it was grown in the dark to become non-chlorophyllous.Although net O₂ evolution could not be detected in the photomixotrophicline throughout the growth cycle when assayed under suboptimumlight intensity, reaccumulation of starch and a marked increasein cell fresh weight upon addition of minerals, vitamins and2,4-D without sucrose at the late logarithmic phase indicatedthe development of photosynthetic activity under the cultureconditions. ¹The investigations reported were included in the thesis submittedto the Graduate School, Faculty of Agriculture, Kobe University,in partial fulfillment of the requirement for M. Agr. degree. (Received May 30, 1988; Accepted October 5, 1988)     

Disposition of Pollen in situ and its Relevance to Anther/Pollen Culture

Disposition of pollen in immature anthers of Hordeum vulgareis illustrated by scanning and transmission electron microscopy.Freeze-fracture confirms that the pollen is confined to a uniseriatecolumn aligned against the tapetum. There is no free pollenin the lumen of the anther loculi. In contrast, in Nicotianatabacum and Paeonia lactlflora the pollen is disposed at randomand occupies the whole of each loculus. Freezing preserves thefluid content of the loculi, appearing in fracture profilesas an amorphous matrix in which the pollen is embedded. Thematrix, which generally obscures the tapetum, is present throughoutthe microspore phase but diminishes as the spores enlarge. Itis still present in N. tabacum and H. vulgare at the first pollendivision, fragmentary at this stage in P. lactiflora, but isno longer discernible in any of the species at the onset ofpollen-grain maturation. Pretreatment of excised Hordeum spikes at 4 ?C during the microsporephase, a prerequisite for anther/pollen culture, disrupts thenormal developmental sequence but does not alter the uniseriatedisposition. Before the spores start to divide, however, thetapetum degenerates and the fluid phase is dispersed. The observations are discussed in relation to isolated pollenculture, float culture of anthers and the switch in programmefrom gametophytic to sporophytic development. Key words: Pollen, Tapetum, Freeze-fracture     

影响小黑杨花药培养中花粉愈伤组织诱导与不定芽分化的几个生理因素

建立了小黑杨花粉愈伤组织与不定芽诱导体系,观察到：（1）25℃和暗培养条件下花粉愈伤组织的诱导率最高（达到89．2％）;（2）25℃、16h·d^-1。光照（光照强度为35μmol·m^-2·s^-1左右）下培养的不定芽诱导率最高（为72.3％）;（3）花芽于0℃下低温储藏时间延长不利于花粉愈伤组织的诱导,而培养早期诱导出的愈伤组织,其不定芽分化能力高于后期诱导的愈伤组织,分化培养基中添加0.1mg·L^-1苯基噻二唑基脲（TDZ）对不定芽分化有促进作用。     

悬浮培养中不同理化因子对烟草发状根生长及烟碱合成的影响

悬浮培养中烟草(Nicotiana tabacum)发状根的生长及烟碱合成受到基本培养基浓度、初始pH值、激素种类和浓度等因素的影响.一个5 cm长的根尖,悬浮在40 mL、pH值为6.0、附加1.0 mg·L-1IAA的1/2 MS液体培养基中,28℃、散光条件下培养30天,烟碱产量可达0.241 mg·mL-1.     

Silver Nitrate Increases Embryo Production in Anther Culture of Brussels Sprouts

Embryo production from anther culture of three poorly respondingBrussels sprouts genotypes was increased in 10 out of 13 experimentsby the inclusion of the ethylene antagonist silver nitrate (AgNO₃)in the medium. When applied to a normally responsive genotype,AgNO₃ increased embryo yield in only one of three experiments.At only one concentration in one of the 16 experiments, didAgNO₃ inhibit embryogenesis. Silver nitrate, anther culture, Brassica oleracea, ethylene     

烟草毛状根诱导及其茄尼醇含量初探

茄尼醇是合成泛醌类药物的重要中间体.以发根农杆菌（Agrobacterium rhizogenes）W.T15834感染烟草叶片诱导产生毛状根,探讨其茄尼醇含量变化.结果显示,获得的毛状根能在无外源生长调节剂的MS固体和液体培养基上自主生长,但在液体培养基中培养的毛状根生长更迅速,也不会形成愈伤组织.甘露碱检测及PCR结果证实,发根农杆菌Ri质粒的rolB基因已在烟草（Nicotiana tabacum）毛状根基因组中整合并得到表达.用改进的HPLC法测定烟草毛状根中的茄尼醇含量,其结果为对照根（种子萌发产生的幼苗根）的1.12倍,但仍比废弃烟叶中茄尼醇含量低43.2%.     

Synergistic Inhibition of Growth by Aluminum and Iron of Tobacco (Nicotiana tabacum L.) Cells in Suspension Culture

Growth of tobacco cells at the logarithmic phase was inhibitedafter treatment with both AlCl₃ and FeSO₄ in modified Murashige-Skoogmedium prepared without P_i and EDTA at pH 5.0 for up to 20 h,whereas cells treated with either AlCl₃ or FeSO₄ (at concentrationsup to 100 µM, respectively) grew normally. These resultssuggest the synergistic inhibition of growth by Al³⁺ and Fe²⁺ions. During the exposure to both salts, there was a lag timeof about 10 h before growth inhibition became apparent in culturesof cells upon treatment with the two salts together. Then theextent of inhibition increased and reached a maximum value afterexposure for 18 h. After the lag period, cells treated withboth salts, but not cells treated with either salt alone, exhibitedsignificant increases in the amounts of both aluminum and ironin the cells and could be stained with hematoxylin, with thenuclei staining especially strongly. Cells treated with bothsalts also exhibited a decrease in the number of cells thatcould be plasmolyzed in 1 M mannitol, a decrease in the numberof cells that could be stained with fluorescein diacetate, adecrease in the amount of potassium in cells, and an increasein the extent of lipid peroxidation. These results suggest thataluminum causes the peroxidation of lipids in the presence ofiron and that lipid peroxidation alters the permeability ofthe plasma membrane and leads to cell death. (Received April 18, 1994; Accepted November 11, 1994)     

Type B Phosphatidylinositol-4-Phosphate 5-Kinases Mediate Arabidopsis and Nicotiana tabacum Pollen Tube Growth by Regulating Apical Pectin Secretion

Phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P₂] occurs in the apical plasma membrane of growing pollen tubes. Because enzymes responsible for PtdIns(4,5)P₂ production at that location are uncharacterized, functions of PtdIns(4,5)P₂ in pollen tube tip growth are unresolved. Two candidate genes encoding pollen-expressed Arabidopsis thaliana phosphatidylinositol-4-phosphate 5-kinases (PI4P 5-kinases) of Arabidopsis subfamily B were identified (PIP5K4 and PIP5K5), and their recombinant proteins were characterized as being PI4P 5-kinases. Pollen of T-DNA insertion lines deficient in both PIP5K4 and PIP5K5 exhibited reduced pollen germination and defects in pollen tube elongation. Fluorescence-tagged PIP5K4 and PIP5K5 localized to an apical plasma membrane microdomain in Arabidopsis and tobacco (Nicotiana tabacum) pollen tubes, and overexpression of either PIP5K4 or PIP5K5 triggered multiple tip branching events. Further studies using the tobacco system revealed that overexpression caused massive apical pectin deposition accompanied by plasma membrane invaginations. By contrast, callose deposition and cytoskeletal structures were unaltered in the overexpressors. Morphological effects depended on PtdIns(4,5)P₂ production, as an inactive enzyme variant did not produce any effects. The data indicate that excessive PtdIns(4,5)P₂ production by type B PI4P 5-kinases disturbs the balance of membrane trafficking and apical pectin deposition. Polar tip growth of pollen tubes may thus be modulated by PtdIns(4,5)P₂ via regulatory effects on membrane trafficking and/or apical pectin deposition.     

Biological and Chemical Induction of Resistance to the Globodera tabacum solanacearum in Oriental and Flue-Cured Tobacco (Nicotiana tabacum L.)

The effects of acibenzolar-S-methyl (ASM) and four combinations of plant growth-promoting rhizobacteria (PGPR) on the reproduction of a tobacco cyst nematode, Globodera tabacum solanacearum, and growth of Nicotiana tabacum (cv. K326 and Xanthi) were tested under greenhouse and field conditions. The PGPR included combinations of Bacillus subtilis A13 with B. pumilis INR7, B. pumilis SE34, B. licheniformis IN937b, or B. amyloliquefaciens IN937a, respectively. Among the four rhizobacterial combinations, IN937a + A13 exhibited the most consistent reduction in G. t. solanacearum cysts under greenhouse and field conditions. No undesirable effects of IN937a + A13 were observed on tobacco growth under greenhouse and field conditions. Use of INR7 + A13 reduced G. t. solanacearum reproduction on flue-cured tobacco cv. K326 but not on oriental tobacco cv. Xanthi. Application of ASM reduced final numbers of G. t. solanacearum cysts, but also resulted in phytotoxicity mainly under the greenhouse conditions. When oriental tobacco seedlings were pre-grown in a IN937a + A13-treated soil-less medium, a single application of ASM at 200 mg/L one week after transplanting significantly reduced G. t. solanacearum reproduction in the field.     

Pollen from cold-treated Nicotiana tabacum buds: Embryogenic capacity,peroxidase activity and partitioning in aqueous two-phase systems

Pollen isolated from fresh and cold treated tobacco (Nicotiana tabacum L. ew. Wisconsin 38) flower buds were separated using aqueous polymer two-phase partitioning and analysed with respect to embryogenic capacity, peroxidase activity and isoperoxidase pattern. Pollen with embryogenic capacity from cold-treated flower buds were enriched in fractions with higher partitioning than those from fresh flower buds, but the amounts were the same. Cold led to a general increase in specific peroxidase activity in pollen fractions enriched in embryogenic pollen, and also to specific changes in the isoperoxidase pattern. Neutral peroxidase species (pI around 7) and alkaline species (pIs around 9) could be related to pollen fractions enriched in embryogenic pollen. The data agree with earlier data showing that the amount of pollen with the potential to form embryos is established at an early stage in flower development, whereas if they really do so depends on how they are pretreated, e.g. by cold treatments of the buds. The latter is also reflected by quantitative and qualitative differences in peroxidase.     

Role of tryptophan residues in a class V chitinase from Nicotiana tabacum

Tryptophan residues located in the substrate-binding cleft of a class V chitinase from Nicotiana tabacum (NtChiV) were mutated to alanine and phenylalanine (W190F, W326F, W190F/W326F, W190A, W326A, and W190A/W326A), and the mutant enzymes were characterized to define the role of the tryptophans. The mutations of Trp326 lowered thermal stability by 5-7 °C, while the mutations of Trp190 lowered stability only by 2-4 °C. The Trp326 mutations strongly impaired enzymatic activity, while the effects of the Trp190 mutations were moderate. The experimental data were rationalized based on the crystal structure of NtChiV in a complex with (GlcNAc)(4), in which Trp190 is exposed to the solvent and involved in face-to-face stacking interaction with the +2 sugar, while Trp326 is buried inside but interacts with the -2 sugar through hydrophobicity. HPLC analysis of anomers of the enzymatic products suggested that Trp190 specifically recognizes the β-anomer of the +2 sugar. The strong effects of the Trp326 mutations on activity and stability suggest multiple roles of the residue in stabilizing the protein structure, in sugar residue binding at subsite -2, and probably in maintaining catalytic efficiency by providing a hydrophobic environment for proton donor Glu115.     

Role of Tryptophan Residues in a Class V Chitinase from Nicotiana tabacum

Tryptophan residues located in the substrate-binding cleft of a class V chitinase from Nicotiana tabacum (NtChiV) were mutated to alanine and phenylalanine (W190F, W326F, W190F/W326F, W190A, W326A, and W190A/W326A), and the mutant enzymes were characterized to define the role of the tryptophans. The mutations of Trp326 lowered thermal stability by 5–7 °C, while the mutations of Trp190 lowered stability only by 2–4 °C. The Trp326 mutations strongly impaired enzymatic activity, while the effects of the Trp190 mutations were moderate. The experimental data were rationalized based on the crystal structure of NtChiV in a complex with (GlcNAc)₄, in which Trp190 is exposed to the solvent and involved in face-to-face stacking interaction with the +2 sugar, while Trp326 is buried inside but interacts with the ?2 sugar through hydrophobicity. HPLC analysis of anomers of the enzymatic products suggested that Trp190 specifically recognizes the β-anomer of the +2 sugar. The strong effects of the Trp326 mutations on activity and stability suggest multiple roles of the residue in stabilizing the protein structure, in sugar residue binding at subsite ?2, and probably in maintaining catalytic efficiency by providing a hydrophobic environment for proton donor Glu115.