Culture of Barley Anthers in Conditioned Media

High yielding anther cultures of Hordeum vulgare cv. Sabarlisare obtained at inoculation densities of 10—20 per mlby use of media previously conditioned by Sabarlis anthers.To achieve these high yields anthers at the mid-unicellularpollen stage (stage 2), stressed in the excised spike for 14d at 7 °C, are necessary, but for conditioning of mediaolder anthers may be used with or without the stress pretreatment.Conditioning for 7 d by anthers at young bicellular pollen stages(stages 5—6) is highly effective. Hormones supplied inthe medium interact synergistically with the conditioning factor. Sabarlis ovaries are shown to be even more effective for conditioningthan anthers, whereas glumes and other parts of the spike arerelatively ineffective. Anthers of oats, rye, wheat, maize,tobacco, and one other genotype of barley are also less effectivethan Sabarlis anthers. The improved method of anther culture is more efficient thanspike culture for the production of pollen callus in Sabarlisbarley.     

Temperature-stress Pretreatment in Barley Anther Culture

Methods of pretreating anthers at different temperatures priorto culture have been tested, with respect to pollen-callus productionand plant regeneration, in Hordeum vulgare cv. Sabarlis. For callus production, pretreatment of excised spikes (in sealedPetri dishes) was more effective than pretreatment of excisedtillers (in water or in polythene) at both 4 and 25 °C.Pretreatment of individual anthers at these temperatures wasdeleterious. Greater callus yields resulted from pretreatmentat 4 than at 25 °C, both for spikes and tillers, 3–5weeks being required for maximal yields at 4 °C and 3–5days at 25 °C. At 4 °C, a shorter pretreatment was requiredfor spikes than for tillers. Pretreatment of spikes was alsomore effective at 4 than at 7, 14 or 20 °C. Pretreatmentof individual spikelets at 4 °C was as effective as thatof whole spikes. For plant regeneration, calluses derived from pretreatment ofspikes were more effective than those derived from pretreatmentof tillers. More plants resulted from pretreatment at 4 thanat 25 °C, both for spikes and tillers. Maximal pretreatmenttimes for plant regeneration generally exceeded those for callusproduction. Following spike pretreatment at 4 °C the maximumfor plant regeneration exceeded that for callus production byabout 2 weeks. With this optimal pretreatment approximately60 per cent of the calluses gave rise to plantlets. Among this60 per cent, for every three calluses giving albinos, two gavegreen plantlets, equivalent to five green plantlets on averagefor every 100 anthers (= two spikes) cultured. The ratio ofgreen to albino plantlets was lower for all other pretreatments. Hordeum vulgare L., barley, anther culture, pollen callus, pollen plant-production, temperature stress     

Anther Culture in Nicotiana tabacum: The Role of the Culture Vessel Atmosphere in Pollen Embryo Induction and Growth

A comparison of four volumes of culture vessel atmosphere revealedthat this variable greatly influenced the induction and growthof pollen embryos from anthers of Nicotiana tabacum. The optimumfrequency of anthers producing embryos and plantlets was foundwith a culture atmosphere of 15 ml per anther, whereas the optimumnumber of plantlets was found with 5.5 ml per anther. A smallvolume (0.5 ml per anther) almost completely suppressed embryoinduction. Removal of specific components of the culture atmosphere(ethylene, carbon dioxide, oxygen) influenced the response ofthe anthers but did not produce a satisfactory explanation ofthe inhibition of pollen embryogenesis by the small cultureatmosphere volume. In particular, the influence of ethyleneabsorption on embryo induction and growth depended both on theculture atmosphere volume and on the stage of development ofthe pollen at the start of culture. Using anthers containingpollen at a stage after the first pollen grain mitosis. ethyleneabsorption was found to increase the survival of induced embryos.Treatment of anthers for 3 d with silver nitrate, a known antagonistof ethylene action, was not an efficient means of increasingthe yield of pollen plantlets.     

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.     

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.     

Development of Pollen Embryoids in Anther Cultures of Datura innoxia: I. GENERAL OBSERVATIONS AND EFFECTS OF PHYSICAL FACTORS

To obtain the maximal production of pollen embryoids in culturedanthers of Datura innoxia, the critical stage of anther developmentand the effect of physical factors, such as the precise modeof implantation of the anthers in the culture medium, light,temperature, and pH, were studied. In almost all media used,anthers containing uninucleate pollen were the best for initiationof embryogenesis. Variations in light and temperature also affecteddevelopment of the embryoids significantly. The percentage ofanthers producing pollen embryoids increased almost linearlywhen the temperature was raised from 22 to 30 °C. At lowertemperatures (15 to 20 °C) no embryoids were produced. Cultureskept in darkness produced embryoids, but upon transfer of culturesto the light the percentage of responding anthers increasedconsiderably.     

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     

Environmental control and evidence for predetermination of pollen embryogenesis inNicotiana tabacum pollen

Summary The effect of daylenght and temperature for the donor plants (Nicotiana tabacum var. Badischer Burley) on the formation of pollen competent for embryogenesis (P-pollen) by the three possible routes (during normal flower developmentin situ (pollen dimorphism), during cold-treatment of excised flower buds, in cultured anthers) was studied. In all three routes, P-pollen frequency (premitotic pollen, before 1. sporophytic division, PPF) was affected in essentially the same way. At 24 °C and long days, PPF was low and short days had only a slightly increasing effect. At 18 °C and long days, PPF was higher and short days further increased it. Correlated with PPF under the different growth regimes was the percentage of units with more than one vegetative-type nucleus (normal embryos + abortive embryos + multinucleate pollen) in 3 weeks old anther cultures. Under greenhouse conditions, PPF was generally higher than at 24° in growth rooms and showed a maximum in the winter months. Plant age did not affect PPF. These results give further evidence that pollen embryogenesis is predetermined before excision and culture of the pollen or anthers.     

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.     

The Influence of Donor Plant Genotype and Environment on Production of Multicellular Microspores in Cultured Anthers of Brassica napus ssp. oleifera

Studies of the effects of genotype and pre-flowering environmentalconditions on the production of multicellular microspores wereundertaken th four highly inbred lines of Brassica napus sap.oleifera. These lines were first grown in shaded and unshadedenvironments at 20/15°C arid unshaded at 30/25°C ina daylight phytotron. Buds were harvested from half the plantswhen first visible in the rosette and later from the remainingplants at the time when the first flower opened. The frequencyof microspores at a specific stage of development varied widelywithin a relatively narrow range of bud lengths. Uninucleatemicrospores were not detected in anthers from buds less than1·5 m or greater than 3·0mm long, but were generallypresent in frequencies of greater than 50 per cent in anthersfrom buds which were between 2·0 and 2·5 mm inlength. However, the bud length at which the highest frequencyof uninucleate microspores was detected varied significantlybetween genotypes and between the environments in which theywere grown. Examination of the remaining anthers from each budafter a period in culture revealed that the proportion of microsporesdeveloping into multicellular units varied greatly with budlength, an increase in frequency of multicellular microsporesbeing associated with an increase in the frequency of uninucleatemicrospores in the uncultured anther. Genotypes differed, however,in respect of the relationship between uninucleate microsporefrequency and production of multicellular units. Although thefrequency of multicellular units was as high as 57 percent,further development was limited and the number of embryoldsformed was low in all cases (<10 per cent). The frequency of multicellular units in pollen samples frombuds of a length in which uninucleate microspore frequency washigh varied significantly with genotype, temperature and lightconditions under which donor plants were grown, and the stageof inflorescence development at which buds were removed. Underconditions most conducive to multicellular unit formation (20/15°C,unshaded), the maximum frequency of multicellular units foreach genotype in buds from young inflorescences ranged from11·5 to 56·5 per cent. Shading or exposure tothe higher temperature was associated with a marked reductionin production of multicellular units. Higher frequencies ofmulticellular units were generally detected in microspore samplesfrom younger inflorescences irrespective of genotype or environment. Two of the four inbred lines were selected for a second experimentin which responses to vernalization and photoperiod durationwere monitored. There was a significant reduction in the numberof leaf nodes formed prior to floral initiation in both genotypesfollowing exposure to vernalization and/or a longer photoperiod,the response to photoperiod being more pronounced. Exposureto 4 weeks vernalization was accompanied by a significant increasein the frequency of multicellular units in both genotypes, thefrequency being double that in unvernalized plants under thelonger photoperiod. By contrast, genotypes differed sharplyin their response to photoperiod. In TB 20, the frequency ofmulticellular units was unaffected by an increase in day lengthirrespective of whether seed had been vernalized or not. Onthe other hand, in TB 42 the frequency of multicellular unitswas substantially greater in the 24 h day than in the 12 h day,being 27·3 per cent vs 13·0 per cent in the caseof unvernalized plants and 66·7 per cent vs 18·2per cent in the case of vernalized plants. Brassica napus, anther culture, pollen embryogenesis, genotype-environment interaction     

Callus induction in culture of Oenothera hookeri and Oenothera picensis anthers

In the present work we try to determine optimum conditions for callus induction in anther culture of Oenothera hookeri and O. picensis. The anther callus yield was increased when the anthers were cultured on modified MS medium supplied with 2 mg dm^-3 2,4-D and 2 mg dm^-3 NAA, in both species. In O. hookeri, best results were obtained when anthers were excised from 7.2 - 9 mm buds at the stage of vacuolated microspores, then pretreated at 4 °C for 2 d and grown under 16-h photoperiod. The response to anther culture of O. picensis was generally very poor compared with that of O. hookeri. The higher yield of calli was obtained when anthers were excised from 6.2 - 8 mm buds at the stage of vacuolated microspores and grown under continuous light. The cold pretreatment of buds decreased anther response in this species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Influence of Genotype, Plant Growth Temperature and Anther Incubation Temperature on Microspore Embryo Production in Brassica napus ssp. oleifera

Eleven F₁ hybrid genotypes of winter rape (Brassica napus ssp.oleifera) were used in a study of induction and growth of microspore-derivedembryos. Plants of each genotype were grown in controlled environmentsat either a constant 15°C or a constant 20°C, both witha 16 h photoperiod. Equal numbers of buds, approximately 2.5mm in length, containing uninucleate microspores were harvestedfrom each genotype and either pretreated (14 d at 4°C) ordissected immediately after harvest. Anthers were cultured onliquid medium based upon that of Murashige and Skoog (1962)and containing 8% sucrose, 0.5 mg dm^–3 naphthylaceticacid and 0.05 mg dm^–3 benzylaminopurine. Anthers fromequal samples of buds were incubated at 35°C for 0, 1, 2or 3 d before transfer to 30°C (21 d) and then 25°C.After a total of 42 d incubation, cultures were scored for thepresence of macroscopic embryos (1–2 mm in length) andfor the presence of anthers containing aborted embryoids whichhad not developed further. The results showed first that bud pretreatment completely inhibitedinduction and secondly that anthers of all genotypes had anabsolute requirement for a 35°C treatment (optimal duration2 d) in order to induce embryoid formation. In the great majorityof genotypes plants grown at 15°C provided more productiveanthers than plants grown at 20°C. However, within eachtreatment there were great differences both in the frequencyof anthers showing induced embryoids and of the final yieldof embryos. There was evidence that hybrids with a common parentresponded similarly under certain treatments. This confirmedthe importance of genotypic control for some components of embryoyield. Key words: Brassica napus, Rape, Anther culture, Pollen, Haploid     

Ethylene Production and Plantlet Formation by Nicotiana Anthers Cultured in the Presence and Absence of Charcoal

Anthers of Nicotiana tabacum produce ethylene when culturedfor plantlet production. The rate is at a maximum 1–2weeks after the onset of culture. Charcoal in the medium increasesthe proportion of androgenic anthers in N. tabacum and severalother Nicotiana species. The level of ethylene in culture vesselsis reduced by charcoal. However, complete removal of ethylenedoes not significantly alter the incidence of androgenesis,nor does continuous flushing of cultures with air. It is concludedthat although charcoal reduces ethylene in the gas phase ofthe cultures its effect on androgenesis is exerted through someother mechanism.     

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     

Influence of Temperature on Potentiation of Cellulysin-Induced Ethylene Biosynthesis by Ethylene

The role temperature plays during ethylene pretreatment on subsequentinduction of ethylene biosynthesis by Cellulysin or by a partiallypurified ethylene inducing factor (EIF) from Cellulysin in tobacco(Nicotiana tabacum L., cv. Xanthi) leaf discs was studied. Leaveswere pretreated with ethylene at three temperatures (7°C,25°C, and 40°C) followed by the induction of ethylenebiosynthesis at 23°C. At 25°C, ethylene pretreatmentstimulated subsequent Cellulysin-, EIF- or ACC-induced ethylenebiosynthesis and conjugation of ACC. At 7°C ethylene pretreatmenthad little effect on subsequent Cellulysin-, EIF- or ACC-inducedethylene biosynthesis, while 40°C pretreatment inhibitedsubsequent Cellulysin-, EIF- or ACC-induced ethylene biosynthesisat 23°C. The high temperature (40°C) pretreatment inhibitedsubsequent conversion of ACC to ethylene in both air and ethylenetreated tissues. (Received May 20, 1988; Accepted January 24, 1989)     

An improved method for dihaploid production in Nicotiana rustica through anther culture

Summary The efficiency of dihaploid production from anther culture in N. rustica has been improved by studying the effects of pretreatment temperature, pretreatment duration and initial anther stage on anther response, anther productivity and time to first plantlet production. Pretreatment was most effective on anthers at or around the stage of pollen mitosis. Pollen mitosis stage anthers pretreated at 9 °C for 15 days gave the best results. Both spontaneous and induced dihaploids were obtained. Small plantlets treated with 0.4% colchicine and 2% DMS solution for 5 h produced the maximum number of dihaploids (more than 50%). These considerable improvements in the efficiency of the techniques have made dihaploidy an attractive method for producing inbred lines in N. rustica. This will permit a large scale comparison of dihaploids with more conventional methods of inbreeding such as single seed descent and pedigree breeding.     

Successful development of a shed-microspore culture protocol for doubled haploid production in Indonesian hot pepper (Capsicum annuum L.)

Various systems of anther and microspore cultures were studied to establish an efficient doubled haploid production method for Indonesian hot pepper (Capsicum annuum L.). A shed-microspore culture protocol was developed which outperformed all the previously reported methods of haploid production in pepper. The critical factors of the protocol are: selection of flower buds with more than 50% late unicellular microspores, a 1 day 4°C pretreatment of the buds, followed by culture of the anthers in double-layer medium system for 1 week at 9°C and thereafter at 28°C in continuous darkness. The medium contained Nitsch components and 2% maltose, with 1% activated charcoal in the solid under layer and 2.5 μM zeatin and 5 μM indole-3-acetic acid in the liquid upper layer. All the ten genotypes of hot pepper tested, responded to this protocol. The best genotypes produced four to seven plants per original flower bud. This protocol can be used as a potential tool for producing doubled haploid plants for hot pepper breeding.     

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     

Improved callus formation and plant regeneration for shed microspore culture in asparagus (Asparagus officinalis L.)

 To establish an efficient asparagus microspore culture system, experiments were conducted to investigate the effects of medium components, period of cold pretreatment for flower buds, and period of anther co-culture on culture response. All factors affected the frequency of asparagus microspore division and the yields of microspore-derived calli. The best results were obtained by pretreating genotype G459 flower buds at 4  °C for 7–9 days, co-culturing anthers with shed microspores for 14 days, and including 6% sucrose, 2 mg l^–1α-naphthaleneacetic acid and 1 mg l^–1 N⁶-benzylaminopurine in the culture medium. After 4 days of culture, most shed microspores contained starch-like bodies and died. The 2% of shed microspores lacking these structures divided to produce microcalli. For the best treatments in the different experiments, about 140 calli per 100 anthers were recovered. Cultured on four different regeneration media, 19.6–21% and 3.9–8.0% of microspore-derived calli produced shoots and embryos, respectively, and ultimately plantlets, among which 49% were haploid, 34% diploid, 4% triploid and 11% tetraploid. Received: 3 September 1998 / Revision received: 25 November 1998 / Accepted: 5 December 1998     

A comparative study of genotypic and environmental response to androgenesis in Nicotiana rustica

Summary A total of six genotypes of Nicotiana rustica comprising the two F₁'s (V₂ × V₁₂ and V₁ × V₅) and their parents were evaluated for their efficiency in haploid production. Excised immature flower buds with pollen at late uninucleate to early binucleate stage were pretreated for 21 days at 5 ° or 7 °C, or for 15 days at 9 °C before culturing on Nitsch's medium+ 0.1 mg/l NAA. The effects of genotype, pretreatment and their interaction were tested on anther response, anther productivity and days to first plantlet formation. Highly significant genotype X pretreatment interaction and differences between genotypes were observed for all three characters. Significant differences between pretreatments were observed for anther productivity only. The performance of V₁₂ both in respect of anther productivity and response was highest whereas that of V₅ was the lowest. Analysis of variance showed that a simple additive genetic model was not adequate to explain the above variation due to significant additive genetic and dominance interactions with the pretreatment.