Effects of High Temperature Stress on Brassica Pollen: Viability, Germination and Ability to Set Fruits and Seeds

Pollen grains of Brassica juncea (L) Czern. were exposed tohigh temperature (45, 60 and 75°C, for 4–24 h) andtheir viability [using the fluorochromatic reaction (FCR) test],germination, and ability to set fruits and seeds were investigated.High temperature up to 60°C did not affect pollen viability;these pollen samples germinated on the stigma and produced fruitsand seeds. There was no correlation between viability and seedset in pollen samples exposed to 75°C; they showed onlyabout 25% reduction in viability but failed to germinate onthe stigma and, consequently, to set seeds. However, these pollensamples induced parthenocarpy. A clear relationship betweenviability and seed-set was observed when the extent of retentionof fluorescence in pollen over a 2-h period was used to assessviability. The fluorescence was retained even after 2 h in pollensamples which induced seed-set, while in those which failedto induce seed-set the fluorescence was lost in about 60 mindue to the leakage of fluorescein. Thus, in stressed pollen,the extent of retention of fluorescence may give a better indicationof pollen function than initial fluorescence, as is being doneroutinely in FCR test. Brassica, White Sim, carnation, Petal senescence, ultrastructure, ethylene, climacteric vacuoles, membranes, wall lysis     

Sinapine Leakage from Non-viable Cabbage Seeds

Seeds leak many compounds during the early phases of germinationand seed viability may be associated with differential leakageof specific compounds. Leakage of a fluorescent compound fromnon-viable cabbage (Brassica oleracca var. capitata L.) wasdocumented and studies were performed to identify the fluorescentcompound. Imbibed samples of both heat-killed (HK) and viablecabbage seeds were submerged in a viscous colloidal gel. After2 h to 4 h, a fluorescent halo was observed under U.V. lightaround the heat-killed seed but not around the viable seed.Viable and HK seeds were imbibed in water for 8 h and the pHof the leachate was adjusted to either 7 or 10. The absorptionspectra of leakage from HK seeds revealed peak values at 322nm and 388 nm at pH 7 and 10, respectively. This pattern wasnot observed from viable seed leakage. Two-dimensional paperchromatography was conducted on the HK seed leachate. Four fluorescentspots were observed after development first with BAW (n-butanol:acetic acid: water, 4: 1: 5 by vol.) followed by 6% acetic acid.One of the fluorescent spots (spot 3) was studied further dueto its observed intensity. Sinapine thiocyanate was preparedfrom rapeseed oilmeal and used as a reference sample. Absorptionspectra of spot 3 and sinapine thiocyanate were similar at bothpH 7 and 10. Spot 3 had identical R_F values using three solventsystems and identical colour reactions in five tests comparedto sinapine thiocyanate. It was concluded that sinapine wasthe major compound responsible for the fluorescent leakage fromHK cabbage seeds. Key words: Leachate, viability, Brassica     

Freezing injury to ovules, pollen and seeds in winter rape

Winter rapeseed (Brassica napus, cv. Samouraï) flowersearly in spring and, under field conditions, short freezingperiods can occur. Unacclimatized plants were freeze-stressed(–3°C for 4 h) at different developmental stages ofbuds, open flowers and seeds. The dissection of pistils from stressed plants showed that freezingresults in shrivelled ovules. We assessed freezing injury onthe basis of per cent of shrivelled ovules: ovule sensitivitybegins early (8 d before anthesis) but increases up to anthesis.Crosspollination of stressed pistils with non-stressed pollenshowed that recording of freeze-injured ovules is a good methodfor early estimation of the effect of stress on seed yields. On the other hand, stress does not reduce the viability of pollen,except when it was applied at the binucleate pollen stage. Useof frozen pollen x nonstressed pistils has little effect onseed yields. Freeze injury on seeds was assessed by seed filling:seeds are very susceptible just after fertilization until 20d after fertilization (DAF). Freezing seems to inhibit seedfilling. A germination test of stressed seeds during their developmentindicated that embryo viability is not affected if the stressoccurs after 35 DAF. As the embryos develop, resistance to stressincreases. Key words: Brassica napus, rapeseed, freeze injury, pollen and ovule, seed filling     

Distribution of {beta}-Thioglucosidase Activity in Intact Plants, Cell and Tissue 6Brassica napus L.

Distribution of myrosinase activity in extracts from seeds,intact plants, cell cultures and regenerated callus and plantsof Brassica napus L. was determined by the rate of glucose formationfrom glucosinolate hydrolysis. Calli with shoots and regeneratedplants were obtained from protoplasts or from explants. Of the seedling organs from Brassica napus L. cv. Niklas, hypocotylsshowed the highest myrosinase activity. In cotyledons a nearlyconstant enzyme activity was determined over the first 6 d,followed by a gradual decline. Roots showed a fast decline inenzyme activity over the investigated period. Freshly-isolated protoplasts contained less myrosinase activitythan the original intact tissue. The enzyme activity in developingcalli generally decreased during the first culture periods.After the initial decline a low activity was found which wasstable for a period of more than 2 years. The enzyme activityshowed fluctuations when measured at different times after mediumchange. Protoplast calli with regenerated shoots showed a considerablyhigher myrosinase activity than calli without shoots. Myrosinaseactivity was also found in explant calli including explant callifrom cotyledons and hypocotyls after induction of shoots. Myrosinase activity in seeds from 21 cultivars of Brassica napus,Brassica campestris, Sinapis alba and Raphanus sativus was testedand the highest myrosinase activity was found in seeds fromthe Sinapis alba cultivar Trico while the lowest activity wasfound in the Brassica campestris cultivar Rapido III. Leaf, stem and inflorescence from flowering regenerated or seed-grownplants contained a low but significant myrosinase activity.In contrast, roots showed a high myrosinase activity. The resultsobtained from regenerated plants indicate that the myrosinasesystem is stable in vitro culture, and that the glucosinolate-myrosinasesystem is active in calli tissue. Key words: Myrosinase (thioglucoside glucohydrolase, E.C. 3.2.3.1), in vitro cultures, intact plants     

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     

Ovule Fertilization and Seed Number per Pod Determination in Oil Seed Rape (Brassica napus)

Ovule fertilization in relation to seed number determinationin spring rapeseed, Brassica napus var. Maris Haplana, was investigated.Failure of fertilization was found to be the major factor limitingthe number of seeds per pod in both the basally and apicallylocated pods on the terminal inflorescence. Sufficient pollengermination on a stigma did not guarantee full seed set andeven when pollen tubes were present at the micropyle regions,ovules were not penetrated. Some seeds aborted: in some, triplefusion did not take place while other seeds were small and deemedat disadvantage to compete for available nutrients. Brassica napus, fertilization, ovules, seeds     

Low Temperature Affects Pattern of Leaf Growth and Structure of Cell Walls in Winter Oilseed Rape (Brassica napus L., var. oleifera L.)

Three-week acclimation of winter oilseed rape (Brassica napusL. var. oleifera L.) plants in the cold (2 °C) resultedin a modified pattern of leaf cell enlargement, indicated bythe increased thickness of young leaf blades and modified dimensionsof mesophyll cells, as compared with non-acclimated tissuesgrown at 20/15 °C (day/night). The thickness of leaf cellwalls also increased markedly during cold acclimation but itdecreased in response to a transient freezing event (5 °Cfor 18 h followed by 6 or 24 h at 2 °C, in the dark). Cellwalls of the upper (adaxial) epidermis were most affected. Theirultrastructure was modified by cold and freezing treatmentsin different ways, as revealed by electron microscopy. Possiblereasons for the cold- and freezing-induced modifications inthe leaf and cell wall morphology and their role in plant acclimationto low temperature conditions are discussed. Copyright 1999Annals of Botany Company Acclimation, Brassica napus var. oleifera, cell wall ultrastructure, cold, freezing, leaf structure, winter oilseed rape.     

Localization of Phenolic Compounds in the Root Cap Columella of Six-year-old Dry Seeds of Brassica napus during Imbibition and Germination

In 6-year-old seeds of Brassica napus the columella cells haveno necroses and resemble in structure the cells of the 2-year-oldembryo. The outermost layer of the columella shows a structuresimilar to that of the lateral region of the root cap, as itcontains protein bodies, rare in layers of the columella closerto the promeristem, which, in turn, contain numerous mitochondriaand plastids. Phenolic compounds in the dry embryo are on thesurface of the root cap in the space between the plasmalemmaand the cell wall, and in small vesicles which presumably remainedfrom degradation of ER. Imbibition promotes further extrusionof phenolics outside the plasma membrane. Long sheets of ERare visible after 9 h imbibition. After 24 h phenolics of moredense structure are localized in some dilated parts of the ER.This suggests that new production of defence compounds startswithin 24 h in water, a few hours earlier than in 2-year-oldseeds.Copyright 1994, 1999 Academic Press Brassica napus, phenolics, root columella, germination     

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     

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     

Alcohol Stress on Soya Bean Seeds

When soya bean seeds were exposed to pure aliphatic alcohols,the shorter alcohols were the most damaging (methanol > ethanol> n-propanol > n-butanol); when the alcohols were appliedin equal volumes of water, the opposite was found (n-propanol> ethanol > methanol), leakage of solutes from the pre-treatedtissue during subsequent imbibition in water was associatedin each case with the loss of germination and a decline in axisgrowth. Damage by the pure alcohols was related to the extentof their penetration and the amount of phospholipid eluted,injury caused by alcohols in the presence of water did not exhibitthese functions. It is proposed that damage to seeds by alcoholsis due to the elution or displacement of cellular phospholipidsand possibly the partial denaturation of membrane proteins Membranedamage is considered to be a prime cause of injury to the seed. Glycine max (L.) Merr., soya bean, seed, denaturation of membranes, alcohols, phospholipids     

Defoliation and its Effects on Pod and Seed Development in Oil Seed Rape (Brassica napus L.)

Both in vivo and in vitro techniques have been used to followthe development of individual pods on the terminal inflorescenceof undefoliated and defoliated plants of oil-seed rape (Brassicanapus cv. Maris Haplona). For any pod, a rapid increase in podlength occurred between 2 d and 8 d after flower opening andthis preceded by approximately 2 d the increase in pod width,the rate of which was less than that for length. An increasein the diameter of individual seeds coincided with the increasein pod width. Regional increases in the length and width ofpods were associated with the presence of developing seeds inthese regions. Key words: Brassica napus L., Development, In vivo, Pod and seed, Stress     

Purification and Characterization of Proteins from the 2S Fraction from Seeds of the Brassicaceae Family

The 2S protein fractions from Brassica rapa, Brassica oleracea,and Brassica napus seeds have been obtained and their componentspurified and characterized. These albumins represent about 13%of the total seed protein extracted with saline buffer. Thecalculated molecular weights of the eleven purified proteinsare very similar, about 14 500 Daltons, although two componentsisolated from B. napus exhibit a lower molecular weight. Theamino acid compositions of the isolated proteins present a seriesof common features: a high content of Cys and basic residuesas well as a very high amount of Pro (15%) and Glx (30%). Theeleven purified proteins cross-react with antibodies againstSin a I, the 2S protein from yellow mustard seeds. The obtainedresults suggest the existence of homology between the 2S albuminsof Brassicaceae seeds. Key words: Brassicaceae, seeds, storage protein     

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.     

Seed Viability Determinations in Cabbage Utilizing Sinapine Leakage and Electrical Conductivity Measurements

A need exists for the development of rapid seed quality teststhat determine viability (defined as the ability to germinate)without the necessity for completion of germination. In cabbage(Brassica oleracea var. capitata L.) the compound sinapine,a choline ester of sinapic acid, has been shown to leak fromheat-killed (HK.) but not from viable seeds. Sinapine leakagewas studied as a more accurate method for identifying viableseeds than the conductivity test. After an 8 h soak, a 26-folddifference in sinapine leakage was detected between HK and viableseeds (as measured by the absorbance at 388 nm at pH 12) comparedto a 4-fold difference measured by electrolyte leakage. Theremainder of the studies were conducted on two seedlots of thesame cultivar. Viability was predicted on the same seed by assessingleakage from individual seeds into the soak water using threemethods; soak water colour, absorbance at 388 nm, and electricalconductivity. This information was compared with the actualgermination of each seed for the two seedlots. For both seedlots,the presence of sinapine in seed soak water (detected eitherby a yellow solution colour at high pH or by high absorbance),identified more seeds that were non-viable than the partitioncoefficient calculated from conductivity measurements (76%,72% and 28% of non-viable seeds were identified, respectively).It is proposed that leakage of sinapine is a better predictorof cabbage seed viability than electrical conductivity. Key words: Seed deterioration, seed leakage, germination prediction     

High Temperature Induced Male and Female Sterility in Canola (Brassica napus L.)

Male and female sterility was induced in canola or rapeseed(Brassica napus L. cv. Westar) flowers grown under high temperatureconditions (32°C/26°C; day/night). At maturity, themajority of flower buds remained closed but had protruding stigmas.The stamens were reduced in size and the anthers showed abnormalmicrosporogenesis. The gynoecia, although normal in appearance,did not set seed and ovule development was aberrant. Flowersof the ogu CMS line of B. napus also showed similar ovular abnormalitiesunder high temperatures and male fertility was not restoredin them. The observations presented suggest that high temperaturesin the field may adversely affect the yield of canola. Temperature, male and female sterility, rapeseed, canola, Brassica napus     

Cytochemical Localization of Phenolic Compounds in Columella Cells of the Root Cap in Seeds of Brassica napus--Changes in the Localization of Phenolic Compounds during Germination

Changes in the localization of phenolic compounds were investigatedin columella cells of embryonic roots in 1-year-old Brassicanapus seeds during 48 h of imbibition and germination. In dry,dormant seeds, phenolic compounds were located in the apoplasticcompartment between the cell wall and plasmalemma in the outermostlayer of the columella. These apoplastic phenolic deposits disappearedduring the activation processes associated with imbibition andgermination, but new deposits appeared successively in the nucleus,ER cisternae, protein bodies and on the outer surface of theroot cap. A large number of phenolic deposits were observedin the outermost part of the columella, becoming less frequenttowards the initial centre. Their appearance coincided withrestoration of the ER and immediately preceded cytological activation.After the primary root had emerged from the seed coat, depositsof phenolic compounds disappeared from the cytoplasm, but simultaneouslyappeared in the vacuoles. Copyright 1999 Annals of Botany Company Brassica napus var. oleifera, root columella, germination, phenolic compounds localization.     

Turgor Maintenance by Osmoregulation inBrassica napusandB. junceaunder Field Conditions

Indian mustard (Brassica juncea(L) Czernjacw) maintains higherleaf turgor than canola (B. napusL.) under water deficits andthis is related to the greater yield of mustard under theseconditions. The work reported in this paper was designed tostudy the way mustard maintains this turgor advantage. It wasbased on three field experiments that each used at least twocultivars or lines of each species. The leaf water potentialat which leaves reached zero turgor was consistently lower inmustard than in canola (up to 1.1 MPa lower). This differencearose from a greater rate of decline in leaf osmotic potentialwith declining water potential in mustard rather than from anydifference in the osmotic potential at full turgor. Calculationsof solute accumulation showed that mustard had a greater capacityto osmoregulate than canola, with this capacity being the basisfor its advantage in turgor maintenance. Other differences inplant water relations were consistent with the differences inosmoregulation, with the predicted relative water content ofleaves at an osmotic potential of -2.5 MPa being 0.43 for canolaand 0.61 for mustard. Mustard's greater capacity to accumulatesolutes is concluded to be a major factor in its greater yieldunder water deficits. Brassica napusL.; Brassica juncea(L) Czernjacw; Indian mustard; canola; water deficit; plant water relations; osmoregulation; osmotic adjustment; turgor     

Endogenous Hormones in Seeds, Germination Behaviour and Early Seedling Characteristics in a Normal and ogura Cytoplasmic Male Sterile Line of Rapeseed (Brassica napus L.)

Endogenous hormones, namely cytokinins (CKs), indole-3-aceticacid (IAA) and abscisic acid (ABA) were quantified by specificenzyme-linked immunosorbent assay (ELISA) in the mature seedof normal (cv. Westar) and ogura (ogu) cytoplasmic male sterile(CMS) lines of rapeseed (Brassica napus L.). Dihydrozeatin (DZ)and dihydrozeatin riboside (DZR) were the major CK base andriboside, respectively, in seeds of both the normal and oguCMS lines. The normal seed had more than 4-fold DZ levels incomparison to that of ogu CMS. On the other hand, the ogu CMSseed had higher levels of CK o-glucosides and CK. nucleotidesthan normal seed. Seeds of the normal line contained 5-foldmore IAA but had one-quarter the level of ABA in comparisonto those of the ogu CMS line. The normal line also had greaterseed diameter and weight than the ogu CMS line and the normalseed germinated earlier than the male sterile seed. DZ (10^–6M) promoted the germination of ogu CMS seeds, but it was notcomparable to that of the normal line. ABA (10^–6 M) inhibitedseed germination of ogu CMS but had little effect on the normalline. The normal seedlings had shorter primary roots, more lateralroots, longer hypocotyls, greater cotyledon fresh weight andhigher chlorophyll levels in comparison to ogu CMS seedlings.Exogenously supplied DZ, IAA and ABA affected the various parametersof both the normal and ogu CMS seedlings, but in most casesdid not fully restore the differences in the two lines. The results presented show that in the ogura cytoplasmic malesterile line of B. napus (1) a number of seed and seedling characteristicsare affected, and (2) the altered seed morphology is accompaniedby changes in the levels of various hormones. Key words: Brassica napus, cytoplasmic male sterility, enzyme-linked immunosorbent assay, hormone, seed germination     

Rapid-cycling Brassica Species: Inbreeding and Selection of Brassica napus for Anther Culture Ability and an Assessment of its Potential for Microspore Culture

A study was made to determine the feasibility of producing,by inbreeding and selection, lines of rapidcycling Brassicanapus L. with high or low potential for anther culture. In contrastto previous observations of B. campestris L., a self-incompatiblespecies, the anther culture potential of the plants of successiveinbred generations of B. napus remained uniform, and antherefficiency was poor, with a maximum of 0.476 embryoids producedfor each anther plated. This negative response to selectionmay have been due to an absence of variation with respect toanther culture ability in the base population, resulting fromthe self-fertility of the species. Cytological studies of culturedanthers of B. napus indicated that in each generation therewas a poor correlation between pollen induction and embryoidproduction. In an attempt to improve the yield of haploid embryoids of B.napus, isolated microspore culture was attempted, and was foundto be at least 60 times more efficient than anther culture,with as many as 32 embryoids being produced from each anther.In experiments designed to ascertain the reasons for such differences,an inhibitory effect of the anther wall on the anther embryogenesisof B. napus was observed, and embryoid yields were improvedby centrifuging buds prior to anther extraction to simulatethe effects of the centrifugation which is a component of themicrospore preparation procedure. Brassica napus, L. anther culture, microspore culture, inbreeding, selection