Storage of Pollen Grains in Organic Solvents: Effect of Organic Solvents on Leaching of Phospholipids and its Relationship to Pollen Viability

In vitro germinability and membrane integrity (as revealed bythe fluorochromatic reaction (FCR) test) of pollen grains ofCrotalaria retusa L. stored in various organic solvents forsix months at –20±2 °C were studied and correlatedwith leaching of lipids, phospholipids, sugars and free aminoacids from pollen grains into organic solvents during storage.Pollen grains stored in organic solvents with low dielectricconstants (a measure of their non-polar nature), such as hexane,cyclohexane and diethyl ether, showed high scores for germinationand FCR and very little leaching of phospholipids, sugars andamino acids. Pollen grains stored in solvents with high dielectricconstants (a measure of their polar nature) such as isopropanoland methanol did not show germination or positive FCR scores,but showed extensive leaching of phospholipids, sugars and freeamino acids. The viability of pollen grains stored in organicsolvents seems to be determined largely by the effect of theorganic solvents on pollen phospholipid composition, which inturn affects membrane integrity and consequently pollen viability. Crotalaria retusa, organic solvents, pollen storage, viability, phospholipids     

Cold-pretreatment of Excised Flower Buds in Float Culture of Tobacco Anthers

Pretreatment of excised flower buds at 7–9 °C is shownto be more effective in float culture of Nicotiana tabacum anthersthan pretreatment at 5 °C. The small temperature differenceresults in greatly enhanced embryo yields when the treatmentis given just before (stage 3), during (4) or just after (5)the first pollen division. Anther productivity (total embryoyield per anther) increases with the time of pretreatment toa peak beyond which further pretreatment is deleterious. Thepeak is attained sooner at 9 than at 7 °C and sooner inanthers pretreated at stage 5 than at stage 3. Peak productivityappears always to be the same. Owing to batch variations, accuratepretreatment-times cannot be specified. Suggested mean timesfor buds of mixed stage are about 12–14 days at 7 °Cand about 7–9 days at 9 °C. Anthers dehisce in culture, the time to dehiscence decreasingwith increasing anther stage. Mid-stage-5 anthers open withinthe first few days, sooner with pretreatment at 9 than at 7°C, and high-yielding free pollen cultures can be obtainedwithout mechanical disruption of the anthers. Buds survive longest when harvested at the beginning of theearly flowering period and kept at 7 °C. It is proposedthat the pretreatment exerts its effect not by altering thecourse of the first pollen division, as suggested by others,but by delaying anther deterioration and thus assuring survivalof a greater proportion of the pollen grains switched into embryogenesis. Nicotiana tabacum L., tobacco, anther culture, embryogenesis     

Virus-Infected Pollen Grains in Paeonia emodi

Tubular virus particles, 25 x 190 nm in length, reacting stronglyin gel diffusion tests and immunoelectron microscopy to tobaccorattle virus antiserum, were detected in pollen grains of aplant of Paeonia emodi. Relative to leaf cells, the pollen grainscontain virus in high concentration, particles being arrangedside by side in long crystalline arrays distributed randomlythroughout the cytoplasm. Presence of the virus is correlatedwith anomalous pollen development and degeneration of mitochondriaand other organelles. The observations are discussed in relationto male sterility and pollen dimorphism in this plant and itsfailure to respond in anther/pollen culture. Key words: Peony, Pollen, Viral Infection     

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.     

Novel 'homeotic' CMS patterns generated in Nicotiana via cybridization with Hyoscyamus and Scopolia

Cytoplasmic hybrids (cybrids) that contain nuclear genetic materialfrom Nicotiana tabacum and cytoplasms from Hyoscyamus nigeror Scopolia carniolica were constructed by protoplast fusions.Both types of hybrids exhibited cytoplasmic male sterility (CMS).Furthermore, unusual floral morphogenesis marked by ‘greenflowers’ deprived of corolla and stamens occurred in themajority of the lines. Backcrosses of these plants with wild-typetobacco demonstrated a maternal inheritance of the ‘greenflower’ trait. After repeated transfer of cytoplasm (‘donor-recipientfusion’) from cytoplasmic hybrid N. tabacum (+H. niger)to albino plastome mutant N. tabacum DSR A15, male sterile tobaccoplants with two types of flowers were recovered (‘greenflowers’ and corolla-containing flowers with transformedstamens). RFLP analysis confirmed that N. tabacum (+ H. niger)and N. tabacum (+S. carniollca) as well as their sexual progeniescontained plastids from H. niger and S. carniolica, respectively.Mitochondrial DNA within the hybrids N. tabacum (+H. niger)originated from H. niger, but was obviously altered. Repeatedparasexual transmission, cybrids in the combination of N. tabacum+N.tabacum (+H. niger), reflected similar characteristics. Cybrids,N. tabacum (+S. carniolica) and their sexual progeny, whichresulted after pollination with wild-type tobacco, containeda modified mtDNA generally originating from tobacco. Furtherhistological analysis established the dramatic difference inthe composition of ‘green flowers’ and flowers ofwild-type tobacco. Therefore, the construction of tobacco cybridswith foreign cytoplasms provides a functional method for thede nova generation of alternative CMS types. Key words: Nicotiana, Hyoscyamus, Scopolia, cybrids, CMS, homeotic patterns     

Nucleic-Acid and Protein Contents of Embryogenic Tobacco Pollen

Anthers of Nicotiana tabacum cv. White Burley containing microsporesin mitosis were cultured for 12-14 d at 23° C in order toinduce mitosis of the vegetative cell (embryogenic grains).After this period the total DNA content of such grains estimatedby gallocyanin-chrome alum photometry was double that of themicrospores in mitosis, whereas the total RNA content was reducedby about one-third. Total protein estimated by naphthol yellowS photometry was approximately the same as at inoculation. Incontrast, total RNA and total protein contents of non-embryogenicgrains in the same anthers were at least four times greaterthan at inoculation. It is suggested that degradation of cytoplasmicinformation concerned in gametophytic differentiation takesplace prior to mitosis of the vegetative cell.     

Shed Pollen Culture in Hordeum vulgare

Anthers of Hordeum vulgare cv. Sabarlis at the mid-unicellularpollen stage, pretreated in the excised spike for 14 d at 7°C, dehisce within 24 h of being floated on the surfaceof liquid medium. About half the pollen (1500 grains per anther)is liberated into the medium. If the anthers are then removedand the cultures re-incubated, calluses develop from the shedpollen in high yields. At low anther densities, 10p–20(1–3 x 10⁴ grains) per ml, medium preconditioned by anthersand supplemented with m-inositol (1000 mg 1^–1) is required,but at high densities, 120 anthers (2 x 10⁵ grains) per ml,preconditioning is less important, the cultured anthers themselveshaving a sufficient conditioning influence. Large-scale dissectionof anthers can be avoided by use of drops of medium, the volumebeing increased gradually as culture proceeds. Pollen remainingin the anthers after 3 d gives rise to calluses if isolatedmechanically and cultured in the inositol medium. The use ofshed pollen is seen as particularly valuable for culture inspecies whose anthers are small, tedious to dissect out anddifficult to process without severe damage.     

Anther Culture of Lolium temulentum, Festuca pratensis and Lolium x Festuca Hybrids. II. Anther and Pollen Development In Vivo and In Vitro

The various pathways of pollen development were investigatedin cultured anthers of Lolium temulentum, Festuca pratensisand the L. multiflorum x F. pratensis hybrid ‘Elmet’.In all three, development from the vegetative cell was the predominantpathway of pollen callus development. However, there were characteristicdifferences in the behaviour of the generative cell. In L. temulentumit remained attached to the pollen wall and degenerated, whereasin F. pratensis it divided. In ‘Elmet’ it detachedfrom the pollen wall and remained undivided. Both polarizedand unpolarized partitioned calluses were observed. Developmentof the fusion product of the vegetative and generative nucleiwere also observed in anthers of L. temulentum. Anomalous grainswere not found to be major source of pollen calluses. Sections of anthers of L. temulentum were used to investigatethe origin of S pollen grains, the small pale-staining grainswhich denote pollen dimorphism. Such grains form out of contactwith the tapetum and are therefore determined before or duringmeiosis (i.e. before harvest of anthers for culture). Sectionswere also used to demonstrate the influence of the durationof pretreatment on the development of the middle layer of theanther wall. Festuca pratensis, Lolium temulentum, Lolium x Festuca, anther culture, haploid, microspore, pollen     

Pollen dimorphism and anther culture in barley

Summary A dimorphism is observed in barley (Hordeum vulgare L., cv. Akka) pollen when stained with acetocarmine from the mid-binucleate stage onwards. The majority of grains have staining cytoplasms, while the remainder have cytoplasms which take up little or no stain (NS grains). The staining dimorphism cannot be detected at the late-uninucleate microspore stage when anthers are normally cultured, but the evidence suggests that the microspores have already diverged at this time and it is the cells destined to become NS grains in vivo that respond in culture to become pollen calluses. Evidence comes from a comparison of the frequencies of NS grains and pollen calluses and from their distribution between and within anthers.     

Biochemical and Ultrastructural Changes in Pollen ofZea maysL. Grown Under Enhanced UV-B Radiation

The influence of ultraviolet-B (UV-B) radiation on the developmentof the male gametophyte was studied inZea maysL. cv. LG12 grownin a growth chamber under PAR light supplemented with UV-B radiationand compared with a second set of plants grown under PAR light.Pollen samples collected from both groups of plants were culturedon germination medium and it was found that UV-B had no effecton pollen germination. Total pollen protein content was notaffected but UV-B absorbing pigments increased. Some ultrastructuralalterations were observed in pollen and pollen tubes, in particularlarge amounts of electron dense deposits were seen throughoutthe cytoplasm and in association with the pollen wall. In maturespikes of UV-B treated plants, anthers retained numerous pollengrains in their loculi while anthers of control plants werealmost empty. UV-B treatment delayed flowering by 2–3d. These results show that UV-B treatment of maize plants interfereswith flowering, pollen ultrastructure and anther maturationeven though pollen germination is unaffected. The significantincrease of UV-B absorbing pigments in pollen grains could representa defence mechanism that enables plants to complete their reproductivecycle.Copyright 1998 Annals of Botany Company Zea maysL., maize, UV-B radiation, pollen.     

Pollen Heteromorphism in Nicotiana tabacum (Solanaceae)

Pollen heteromorphism is defined as the production by a single plant of different fertile pollen types in all its anthers, and thus all flowers, throughout its life cycle. Eight cultivars of Nicotiana tabacum, as well as its ancestors (N. tabacum is an amphiploid hybrid 4 × from a cross between N. sylvestris and N. tomentosiformis) and recent hybrids were analyzed. Most cultivars and the hybrids are heteromorphic (producing 3- and 4-aperturate pollen grains), whereas both parent species are homomorphic (3-aperturate). Heteromorphism is a common consequence of polyploidization and these results agree with this interpretation. There is a significant variation in the proportions of the two pollen types among cultivars (genetic component), but also (with a much lower component of variance) within each cultivar, between plants (genets), flowers of a plant, and even anthers of a flower. This is interpreted as a release of the selective pressure: the cultivars of N. tabacum were obtained after several generations of selfing and are themselves selfers. Selfing, by removing pollen mixtures on a stigma, removes pollen competition, which is the drive for heteromorphism, and allows for a large variation of the proportions of the different pollen types.     

Azetidine-2-carboxylic Acid and Lily Pollen Tube Elongation

Azetidine-2-carboxylic acid (AZC), which occurs naturally inLiliaceous plants, is reported to be a proline (pro) analoguePlant cell walls contain ‘extensin’, which is richin hydroxyproline (hyp). Peptidyl hyp arises through hydroxylationof peptidyl pro followed by glycosylation (arabinose attachment)of hyp Because AZC replaces peptidyl prolyl residues, it maybe a useful tool for evaluating the significance of hyp-o-arabinoselinkages in cell elongation. Therefore, we determined the effectof AZC on [¹⁴C]pro uptake, incorporation and conversion to wall-bound[¹⁴C]hyp in relation to elongation of lily pollen tubes whosewalls consist, in part, of hyp-containing glycopeptides TheAZC suppressed pollen germination 9–42 per cent (1–10mM) and subsequent tube elongation 40–54 per cent (0·1–1mM without affecting respiration In contrast, similar hyp concentrationswere without effect on tube elongation Whereas uptake of [¹⁴C]prowas 16·5–6·2 per cent of the control at0·1–1 mM AZC, [¹⁴C]leucine uptake was 85–25per cent of the control. Light microscope radioautography revealedfewer silver grains over tubes elongated in 0·1–1mM AZC than in its absence. Incorporation of [¹⁴C]pro into tnchloroaceticacid (TCA)-precipitable cytoplasm was reduced by only 10 percent at 0·01–1 mM but 43 per cent at 10 mM AZCGel filtration of cytoplasm from pollen germinated without AZCbut with [¹⁴C]pro resulted in labelled void volume (V) and threeretarded peaks (RI–III) Incorporation into V and RI wasinhibited at both 0·01 and 1 mM AZC These AZC concentrationsreduced conversion of [¹⁴C]pro to wall-bound hyp by 20 percent However, total incorporation of [¹⁴C]pro into salt-water-purifiedwall fractions was suppressed 47–53 per cent (0·1–1mM AZC). Lilium longiflorum, lily, hydroxyproline, proline, azetidine-2-carboxylic acid, pollen, pollen tube elongation     

Experimentally Synthesized Plant Chimeras 2. A Comparison of In vitro and In vivo Techniques for the Production of Interspecific Nicotiana Chimeras

In vitro and in vivo techniques were compared for synthesizingchimeras between Nicotiana glauca Grahm and N tabacum L Interspecificchimeral callus, produced from mixed callus cultures in vitro,was placed on media which favoured only N tabacum shoot formationNone of the 474 regenerated N tabacum shoots incorporated Nglauca cells into their meristems When chimeral callus was regeneratedunder hormonal conditions favouring simultaneous organogenesis,of 397 shoots, only non-chimeral shoots of both species aroseIn vivo, reciprocal splice grafts between species were decapitatedjust above the graft union and treated with or without auxin—lanolinpastes Auxin increased callus formation but inhibited adventitiousshoot formation Three of 209 adventitious shoots arising fromthe graft union were interspecific mericlinal chimeras whichwere later stabilized as periclinal chimeras All three chimerasformed when N glauca was the understock Two of the chimerasarose on untreated shoots which produced no visible callus,indicating that excessive callus formation may be unnecessaryfor multiple cell origin of adventitious shoots to occur Chimeras, tobacco, Nicotiana glauca, Nicotiana tabacum, tissue culture, graft chimeras, callus cultures     

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)     

Pollen Diffusates of Crotalaria retusa and their Role in pH Regulation

Details of the release of proteins and amino acids from culturedpollen grains and the role of the leached metabolites in pollengermination, pollen tube growth and regulation of pH of theculture medium in Crotalaria retusa have been investigated.In unbuffered media, satisfactory pollen germination and tubegrowth occurred over a wide range of pH values 4.0–9.0.This was related to the ability of pollen diffusates to shiftthe pH to 6.25 in all these media. Similar pollen germinationand pH shift was observed when the pollen was eluted twice beforeculturing. When the pH shift was reduced by using buffered media,optimal germination and tube growth occurred only at pH 6.0.Pollen diffusates had a strong buffering capacity. Proteinsand amino acids released from pollen do not seem to have a directrole in pH regulation. The components involved in pH regulationmay originate from the pollen wall as well as from the cytoplasm. Crotalaria retusa L, pH regulation, pollen diffusates, pollen germination     

β-Glucuronidase gene and green fluorescent protein gene expression in de-exined pollen of Nicotiana tabacum by microprojectile bombardment

 Gene constructs containing the β-glucuronidase (GUS) gene or green fluorescent protein (GFP) gene under the control of pollen-specific promoter Zm13-260 from maize were introduced by particle bombardment into de-exined pollen of Nicotiana tabacum. The de-exined pollen exhibited transient expression of the GUS or GFP gene as indicated by histochemical and fluorescent assay, respectively. The frequency of de-exined pollen transformation with the GUS or GFP gene was approximately 6 and 3 times higher, respectively, than that of pollen with intact walls, indicating that pollen deprived of the exine barrier responded better to foreign gene transfer than did the original. Cytological observation of GUS-expressing pollen grains showed that introduced gold particles were visible in the cytoplasm and vegetative nucleus as well as in the generative nucleus. GFP-expressing pollen tubes were observed in the style even after pollination. Received: 28 October 1997 / Revision accepted: 13 April 1998     

Effect of Pollen Protein Diffusates on Germination of Eluted Pollen Samples of Petunia hybrida in vitro

Germination in vitro of pollen grains of Petunia hybrida L.is sharply reduced by brief elution with cold distilled water.If eluted substances are added back to eluted pollen germinatingin vitro, the germination capacity is significantly restored.A heat-labile protein fraction (50000–100000 daltons)is responsible for restoring the germination ability. Petunia hybrida L, pollen, protein, diffusates, germination     

Androgenic Stimulating Factors in the Anther and Isolated Pollen Grain Culture of Datura innoxia Mill

Flower buds, anthers, and/or pollen grains collected at thetime of first haploid mitosis and 1–2 d before or afterthis division were submitted to different treatments beforeculturing anthers or isolated pollen grains. In the case ofanther culture, the percentages of androgenic anthem were notedat the end of 2, 3, 4, and 5 weeks of culture. Statistical studiesof the results thus obtained showed that some factors were highlyeffective in favouring androgenesis. The best results were obtained1–2 d after the first haploid mitosis with anther's centrifugedat 40 g for 5 min after cold treatment of the flower buds (48h at 3 °C); these treatments increased the percentage ofandrogenic pollen grains 12-fold. In case of isolated pollengrains, a system of culture particularly favourable for inductionand development of androgenic embryos was found. This systemincluded a cold treatment of the flower buds (48 h at 3 °C),the centrifugation of the isolated pollen grains (120 g for15 min), and culturing them for 20 d in the dark and then incontinuous light.     

In vitro pollen embryogenesis in Nicotiana tabacum L. and its relation to pollen sterility,sex balance,and floral induction of the pollen donor plants

Pollen sterility, sex balance, and floral induction of the pollen donor plants were tested for a possible relation to embryogenesis from in vitro cultured tobacco pollen (Nicotiana tabacum L. var. Badischer Burley). The pollen grains destined to become embryos in culture (P-grains) were sterile for the donor plants as judged by their staining reaction with acetocarmine and fluorescin-diacetate, and by an in vitro germination test. They were produced in high frequency in flowers which exhibited a shift in sex balance towards femaleness. Sex balance could be measured by the relative length of pistil to stamens. High P-grain frequency, high pollen sterility, and a shift in sex balance towards femaleness could be induced by raising the donor plants under short days and/or low temperature (18–15° C) as compared to long days at 24° C. Short days and/or low temperature also reinforced floral induction, revealing that the tobacco variety Badischer Burley is a quantitative short day and low temperature plant and that the variety follows the rule that conditions of strong floral induction shift sex balance towards femaleness. At 12° C and short days, contabescent flowers were formed with completely sterile anthers containing a few and mostly collapsed P-grains. Based on these results, it is now possible to predict conditions by which haploids via pollen embryogenesis might be produced in high frequency from low-yielding and recalcitrant species.Abbreviations DPF dead pollen grain frequency - LD24 long days at 24° C - PD pollen dimorphism - P:S ratio of pistil to stamen length - SD15 short days at 15° C     

Fertility of Deep-frozen Maize (Zea mays L.) Pollen

On average, 50 per cent of maize pollen grains can be kept viableand almost 30 per cent remain fertile for up to a year whenthe water content on a fresh weight basis is reduced to about30 per cent of the original and the pollen is stored at –76or –196 °C. Over this period no significant differencewas found between storage at either temperature. Zea mays L., maize, pollen, fertility, viability