Rapid-cycling Brassica Species: Inbreeding and Selection of B. campestris for Anther Culture Ability

A study was made to determine the feasibility of producing,by inbreeding and selection, lines of rapid-cycling Brassicacampestris with high or low potential for anther culture. Sincethe base population of rapid-cycling B. campestris is self-incompatible,inbreeding was achieved by a combination of bud-pollinationand the application of pollen to the cut surfaces of decapitatedstigmas. Three inbred generations were raised, and in each generationplants were selected for high or low potential for anther embryogenesis.The proportion of viable pollen present in anthers, as indicatedby a fluorochromatic reaction and a germination test, was alsodetermined at each stage. Lines of rapid-cycling B. campestriswith clearly defined high or low potential for anther embryogenesiswere isolated in these experiments. Within each line, however,continuous variation was always observed. Pollen viability andanther efficiency were not correlated. Although inbreeding depressioncaused a significant decrease in pollen viability over the threegenerations, there were no obvious deleterious effects on antherefficiency. In general, over the three generations of inbreeding,no segregation in plant morphological characters was observed,although many developmental abnormalities were seen in the 3rdinbred generation and there was a marked reduction in the numberof seeds set. No association between plant vigour and high orlow anther efficiency was noted. All plants regenerated fromanther embryoids of rapid cycling B. campestris were haploid.By treating anther-derived embryoids, axillary buds and wholeplants with colchicine, dihaploid plants were produced, butthese failed to set seed after self-pollination. The diploidnature of the plants was confirmed, however, when they producednormal seeds after cross-pollination with plants of the basepopulation. Brassica campestris, anther culture, inbreeding, selection     

Haploid Culture and UV Mutagenesis in Rapid-cycling Brassica napus for the Generation of Resistance to Chlorsulfuron and Alternaria brassicicola

The effect of ultra violet (UV) irradiation on cultured isolatedmicrospores of rapid cycling Brassica napus was investigated.The microscpores were highly sensitive to UV, with the calculatedLD₅₀ being an exposure of 20 s. Viability tests suggested thatdeath of the microspores was not immediate, but occurred duringsubsequent incubation (7 d). None of the embryos produced followingUV-irradiation of microscpores showed gross morphological variation.A large number of regenerants was established from embryoidsand grown to flowering. These plants set fertile seed afterselfing. The progenies were assessed for resistance to Alternariabrassicicola and a small number showed increased resistanceto the pathogen, suggesting the generation of novel heritableresistance to this pathogen. In vitro selection revealed heritableresistance to the herbicide ‘Glean’ (active ingredientchlorsulfuron). Brassica napus, microspore culture, UV light, mutagenesis, selection, disease resistance, herbicide, Alternaria brassicicola     

Induction and Growth of 'Microspore-Derived' Embryos of Brassica napus ssp. oleifera

Three cultivars of spring rape (Brassica napus ssp. oleifera),Tower, Willi and Duplo, were used for a study of induction andgrowth of ‘microspore-derived’ embryos, Buds, 2.0mm in length, containing uninucleate microspores were harvestedand stored for 14 d at 4 ?C in darkness. Anthers were then removedand cultured on a liquid medium based upon that of Murashigeand Skoog and containing 8% sucrose, 0.5 mg l^–1 naphthylaceticacid and 0.05 mg l^–1 benzylaminopurine. Cultures werepre-incubated at 35 ?C for 0–3 d and then incubated at30 ?C. After a total of 42 d incubation, cultures were scoredfor the presence of macroscopic embryos (1–2 mm in length)and for the presence of anthers containing abortive embryoidswhich had not developed further. The cultivars differed greatly in terms both of the frequencyof anthers showing induced embryoids and of the final yieldof embryos. Tower showed the highest frequency of induction(maximum 38% of cultured anthers with induced embryoids) whereasthe highest yield (equivalent to 1.1 embryo per cultured anther)was obtained from anthers of the cv. Duplo after a 3 d treatmentat 35 ?C. Yields from the other cultivars were much lower andwere relatively unaffected by the 35 ?C treatment. Key words: Brassica napus, Rape, Anther culture, Pollen, Haploid     

Role of Sucrose in Microspore Embryo Production in Brassica napus ssp. oleifera

Dun well, J. M. and Thurling, N. 1985. Role of sucrose in microsporeembryo production in Brassica napus ssp. oleifera.—J.exp. Bot. 36: 1478–1491. One cultivar of winter oil seed rape (Brassica napus ssp. oleifera)and three cultivars of spring rape were used in a study of theeffects of sucrose on microspore survival and embryo inductionin cultured anthers. A preliminary study on the winter cultivar(Fiona) revealed that the osmotic pressure of the supernatantof anther homogenates was equivalent to a solution of 17% sucrose.A study of microspore survival and embryo induction in thiscultivar on media containing either 8 %, 12%, 16% or 20% sucroserevealed the highest survival (after 16 d) and the greatestnumber of anthers with induced embryos (after 42 d) occurredon the highest sucrose concentration. A subsequent study on three spring cultivars (Willi, Duplo andTower) examined microspore survival at 8 d and embryo induction(42 d) on media containing either 8 % or 16 % sucrose and againrevealed much higher survival and induction at the higher concentration.The variation in response between the cultivars was also reducedby culture at the higher sucrose concentration. The beneficialeffect of the 16% level occurred regardless of the growth environmentof the donor plants and of the stage of pollen development atthe start of culture. However, macroscopic embryos emerged onlyfrom anthers on the 8 % sucrose concentration, suggesting thattransfer of anthers from a high to a normal sucrose concentrationduring culture would ensure that full advantage was taken ofthe much higher initial survival on the higher concentration Key words: Brassica napus, sucrose, microspore embryo production     

Differences in Osmoregulation in Brassica species

Brassica carinata L (cv Carinata-2) and Brassica napus L (cvHNS-3) were tested for osmoregulation under three sets of environmentsOsmoregulation was found to vary markedly between two species,with the cv Carinata-2 having a greater degree of osmoregulationthan the cv HNS-3 Furthermore, the differences in osmoregulationwere related to leaf diffusive conductance and grain yield inBrassica species Thus, it has potential use as a selection criterionin Brassicas Brassica carinata L, Brassica napus L, osmoregulation, relative water content, leaf water potential, turgor potential, osmotic potential, leaf temperature, leaf diffusive conductance     

Cold pretreatment enhances microspore embryogenesis in oilseed rape (Brassica napus L.)

Stress is an essential component during embryogenesis induction in microspore culture. Cold pretreatment has been used in cereal microspore culture but very seldom attempted in Brassica microspore culture. The effect of cold pretreatment of flower buds subjected to a liquid medium on microspore embryogenesis was investigated in spring and winter Brassica napus, as well as in B. rapa and B. oleracea. Cold pretreatment significantly enhanced microspore embryogenesis (by 1–7 fold) compared to commonly used microspore culture protocol in B. napus, while it was less effective in B. rapa or even negative in B. oleracea. The appropriate duration of cold pretreatment was found to be 2–4 days, which stimulated the best microspore embryogenesis. Cold pretreatment was also able to promote embryo development including the improvement of embryo quality and acceleration of embryogenesis. When incorporating with medium refreshing, cold pretreatment could initiate the most microspore embryogenesis than any other treatment used. With further improvement cold pretreatment method may have a positive potential in Brassica breeding programmes.     

Reproductive Development in Oilseed Rape (Brassica napus cv. Bienvenu)

The reproductive development of oilseed rape (Brassica napusL. cv. Bienvenu) was studied using light and scanning electronmicroscopy. By using the two techniques on comparable samples,internal events such as microspore development were relatedto the morphology of the developing floral parts, and this inturn was linked to the growth stage of the plant. Reproductive development, oilseed rape, Brassica napus, growth stage     

甘蓝型油菜游离小孢子培养的胚胎发生

以生长在非控温控光下的４个冬性甘蓝型油菜（ＢｒａｓｓｉｃａｎａｐｕｓＬ．）品种为供体,进行游离小孢子培养。研究发现,多数品种在开花３－７天取材最为适宜。在甘蓝型油菜小孢子培养中只有单核晚期的小孢子才可能发育成胚状体,而花药培养时处于单核早期的小孢子易于发育成胚状体。在适当花期选取发育比较一致的单核晚期小孢子培养,经数小时后,部分小孢子便开始膨大,这是小孢子发育成胚的最早标志,膨大的小孢子中,有部分形成多细胞球并进一步发育成胚。用春性甘蓝型油菜为材料进行蔗糖浓度的实验结果表明：培养３天后,在１６％蔗糖培养基中存活的小孢子最多,达１６．１３％;培养３０天后,胚状体诱导频率则以１３％蔗糖浓度为最高,每花蕾可达１４４个胚状体。如果在１６％蔗糖培养基中培养３天后,添加等体积的１３％蔗糖培养基,能够大大提高胚状体的诱导频率,为仅用１３％蔗糖培养基培养的３．７倍。这一实验体系正在用于抗菌核病的诱变与筛选,并作为外源基因导入的实验体系。     

Comparative Morphological Study of Zygotic and Microspore-derived Embryos ofBrassica napusL. as Revealed by Scanning Electron Microscopy

A comparative morphological study of microspore-derived (MD)and zygotic embryos ofBrassica napusL. was conducted, illustratingsubstantial similarities in external morphology of these embryosthroughout their development. Haploid embryos were producedfrom isolated microspores cultured on high molecular weightpolyethylene glycol (PEG), replacing sucrose as an osmoticum.Morphological changes during the time-course of microspore embryodevelopment induced on PEG (25%) and sucrose (13%) are describedin detail as revealed by scanning electron microscopy (SEM)and compared to the corresponding stages of zygotic embryosdevelopedin ovulo. At the heart, torpedo and early cotyledonarystages, microspore-derived (MD) embryos on PEG closely resembletheir zygotic counterparts. In contrast, the external morphologyof embryos induced on high sucrose medium differs from thatof PEG and zygotic embryos indicating that a high concentrationof sucrose in culture has a morphogenetic effect on MD embryodevelopment inB. napus. Fragments of the original pollen wallare regularly observed at the root pole region and at the tipsof suspensors in MD embryos throughout their development. Thissuggests that polarity in MD embryos might originate from structurallypolarized late uninuclear microspores and early bicellular pollen.Copyright1998 Annals of Botany Company Brassica napusL., scanning electron microscopy, microspore-derived embryo, zygotic embryo, morphology, microspore, suspensor, exine, sucrose, polyethylene glycol.     

Establishment of Isolated Root Cultures of Brassica Species and Regeneration from Cultured-root Segments of Brassica oleracea var. italica

Isolated root cultures which could be maintained over severalmonths by serial subculture were established from Brassica oleraceavar. italica cv. Green Comet F₁. A modified White's medium wasfound to be the best of several salt compositions tested. Theeffects on isolated root growth of the following were also examined;nutritional components, culture vessel type and closure, pHof the medium and auxin type and concentration. Using a mediumdevised for Green Comet, root cultures were established fromsix other B. oleracea, B. napus and B. campestris cultivars. It was possible to regenerate shoots from segments of culturedroots by incubation on agar-solidified media containing cytokininand auxin. The effects on regeneration of various auxins andcytokinins were investigated; the combination of Picloram withKN gave the highest frequency of shoot formation. It was demonstratedthat isolated roots retained their regenerative ability overa period of 5 months in culture. Brassica oleracea var. italica, Brassica napus, Brassica campestris, isolated root culture, shoot regeneration, organ culture     

In vitro Shoot Regeneration from Seedling Root Segments of Brassica oleracea and Brassica napus Cultivars

Root segments taken from aseptically-grown seedlings of Brassicaoleracea var. italica cv. Green Comet were used in an investigationof factors affecting in vitro regeneration. Shoot regenerationwas found to increase with seedling age and to be highest inroot segments adjacent to the hypocotyl and lowest in segmentsadjacent to the root tip. In a comparison of a range of mediaand agar concentrations shoot formation was favoured by complexmedia containing reduced nitrogen and was higher on gelled mediathan in liquid medium. The effects of various cytokinins andauxins were investigated; KN was the best cytokinin and IAAand Picloram the best auxins for shoot induction. Root segmentsfrom six other Brassica cultivars were grown on the medium devisedfor Green Comet; shoots were regenerated from two B. oleraceacultivars and two B. napus cultivars, but not from the B. campestriscultivars tested. Brassica oleracea var. italica, Brassica napus, Brassica campestris, seedling root culture, shoot regeneration     

In vitro microspore selection in maize anther culture with oxidative-stress stimulators

Summary. In order to produce doubled-haploid maize plants tolerant of oxidative stress, in vitro microspore selection was carried out in anther culture with reactive oxygen species (ROS) progenitors such as paraquat, menadione, tert-butylhydroperoxide (t-BHP), and methionine combined with riboflavin. All the ROS progenitors reduced the anther induction, the formation of microspore-derived structures, and their regeneration potential. Abnormal cell divisions and progeny cell degradation could be observed during the development of microspores treated with ROS progenitors. Menadione and t-BHP influenced the microspore developmental pathway, as menadione induced the formation of embryoids, while t-BHP increased the proportion of calli in the microspore-derived structures. As the result of in vitro selection, 15, 10, 10, and 3 fertile doubled-haploid plants were obtained in cultures treated with paraquat, t-BHP, methionine combined with riboflavin, and menadione, respectively. Correspondence and reprints: Agricultural Research Institute, Hungarian Academy of Sciences, Brunszvik utca 2, 2462 Martonvásár, Hungary.     

Influence of Preculture Variables on Microspore Embryo Production in Brassica napus ssp. oleifera cv. Duplo

The effect of four preculture variables on microspore embryoinduction and growth were examined: (1) the source of the budselected for culture (apical or axillary inflorescence; (2)the method of harvest (single harvest of whole inflorescenceor sequential harvest of individual buds; (3) the length ofthe bud (2, 3 or 4 mm); and (4) the application of a 4 ^°Cpretreatment to the bud after harvest. Microscopic and macroscopicanalysis of every anther used for culture permitted an assessmentof the following parameters: (1) the percentage of induced buds;(2) the number of induced anthers per induced bud; (3) the numberof productive buds (with macroscopic embryos) as a percentageof the induced buds; (4) the degree of induction per inducedanther (an estimate of the number of microspores in which initialembryogenic divisions had commenced); and (5) embryoid survival(the number of embryos as a proportion of the degree of induction). The product of parameters 1 and 2 gave the number of inducedanthers and all five parameters were components of the finalyield - the number of embryos produced per bud cultured. It was found that the maximum number of induced buds (67·0per cent) occurred with 2 mm sequentially harvested non-pretreatedbuds. Overall, the values decreased with increasing bud lengthand were lower for pretreated and axillary buds. In contrast,the two other estimates of induction - number of induced anthersper induced bud and degree of induction per induced anther -both had maximum values from 3 mm sequentially harvested, pretreatedbuds from apical inflorescences. The highest final yield ofembryos per cultured bud (44·9) was found with 2 mm non-pretreatedbuds taken from a single harvest of the apical inflorescence.The study therefore confirmed that the different componentsof the final embryo yield are differentially affected by thefour preculture variables tested. These variables must be controlledif reproducible results are to be achieved. Brassica napus, tape, anther culture, pollen, microspore, haploid     

小麦的小孢子培养

小孢子培养是在花药培养的基础上发展起来的一种高效再生体系, 其在植物育种中的作用一直受到关注。但是, 由于培养技术的限制, 小麦(Triticum aestivum)小孢子培养还未能推广应用。近年来小麦小孢子培养发展很快, 展现出广阔的应用前景。     

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     

Molecular Cytogenetic Localization and Characterization of 5S and 25S rDNA Loci for Chromosome Identification in Oilseed Rape (Brassica napus L.)

Chromosome identification in oilseed rape (Brassica napus L.)is extremely difficult using conventional cytogenetic techniquesbecause amphidiploid Brassica species possess numerous verysmall chromosomes with few cytogenetic landmarks. In combinationwith methods for improved chromosome preparations, we used asimplified fluorescencein situ hybridization (FISH) techniqueto localize simultaneously the gene families coding for 5S and25S rDNA in B. napus. The resulting hybridization patterns enabledten of the 19 oilseed rape chromosome pairs to be unequivocallyidentified. Copyright 2000 Annals of Botany Company Brassica napus, oilseed rape, rDNA, molecular cytogenetics, FISH, chromosome identification     

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     

Genetic selection and liquid medium conditions improve the yield of androgenetic plants from diploid potatoes

Summary Solatium tuberosum L. diploid strains with superior androgenetic capacity have been selected for from androgenetic progenies of unselected diploid material. The paper also demonstrates that the use of a liquid medium for culturing potato anthers, instead of the conventional solid agar plates, improves the yield of androgenetic embryoids. The new method, associated with two successive cycles of selection for superior androgenetic response, allows the induction and regeneration of microspore derived plants on a large scale. The best genotype (clone 21 in this paper) regenerates androgenetic plants with a frequency around 30 per each anther plated. Over 80% of the regenerated plants are diploid. It is suggested that the androgenetic embryoids mainly originate from unreduced microspores by a mechanism which maintains a heterozygous or a partly heterozygous genetic situation.     

Modification of cell development in vitro: The effect of colchicine on anther and isolated microspore culture in Brassica napus

In an attempt to discover the biological basis of microspore derived embryogenesis, the effect of the antimicrotubule agent colchicine on anther and free microspore embryogenesis was investigated. The microtubule inhibitor colchicine promoted embryogenesis from cultured anthers, both with regard to the number of anthers responding and the number of embryos being produced per anther. A similar promotional response was also observed with cultured microspores. Although the parameters for cultured anthers and free microspores differed, administration of the drug for a short period immediately prior to pollen mitosis I seems to exert the maximum promotional effect. Of the five cultivars of Brassica napus studied, all responded to colchicine treatment. However, the drug did release more embryogenic potential in poor-responding varieties (i.e. Lirawell and Optima) than in the highest responding variety (Topas). Colchicine also resulted in increased embryogenic response in microspores cultured at lower temperatures.These results are considered in terms of models proposed to explain the switch in microspore development from a gametophytic to a sporophytic pathway. The use ofcolchicine as agent to promote embryogenesis in previously recalcitrant species other than Brassica is also discussed.     

Inhibitory effects of filter-sterilized media on microspore development and embryogenesis in <Emphasis Type="Italic">Brassica napus</Emphasis> and <Emphasis Type="Italic">Triticum aestivum</Emphasis>

Summary Plant tissue culture media, sterilized through commercial filter membrane units, inhibited plant cell growth and development. Embryogenesis was suppressed in microspore cultures of Brassica napus and anther cultures of Triticum aestivum, and in vitro pollen development was suppressed in B. napus. Inhibition of growth and development occurred on media that had been filter-sterilized in low volumes through non-washed filter membrane units of all brands tested, including Nalgene, Corning, Millipore, and Sartorius membrane products. Similar results were obtained with Costar Transwells and Millipore inserts (Millicell-CM.-HA, and-PCF). These deleterious effects were eliminated by washing the membranes with water followed by a small volume of the medium. Media sterilized by filtration through washed filters produced significant improvements of growth and development of embryos and pollen.