Localization of Phenolic Compounds in the Covering Tissues of the Embryo of Brassica napus (L.) during Different Stages of Embryogenesis and Seed Maturation

During embryogenesis and maturation of an embryo the tissuescovering it produce phenolic compounds the localization of whichchanges during maturation of the embryo. In the ovary containinga globular embryo, phenolics are located in the epidermis ofthe integumentum externum and the innermost layer of the integumentuminternum. In the ovule at the stage at which heart- and torpedo-shapedembryos are present, phenolic compounds are visible in the stellarcells, the innermost cells of the integumentum internum andthe endosperm. In hard, green seeds, after the integumentuminternum and layers over the stellar cells gradually disappear,the remaining tissue contains cell walls impregnated with phenolics.Mature, black seeds contain only one distinct layer of cells—stellarcells, which, like the other compressed cell walls, are impregnatedwith phenolics. In this way they constitute a barrier betweenthe embryo and its environment.Copyright 1994, 1999 AcademicPress Brassica napus, seed coat, integumentum, phenolic compounds     

重金属对油菜种子萌发和胚根生长的影响

分析了Hg2 、Cd2 、Ni2 、Co2 、Zn2 5种重金属离子对油菜种子萌发和胚根伸长的影响,以及金属离子K 、Mg2 和Ca2 与重金属的交互作用。结果表明:(1)重金属对油菜种子萌发的抑制作用依次为Hg2 >Cd2 和Co2 >Ni2 >Zn2 ,而对胚根生长的毒害作用依次为Hg2 >Cd2 >Co2 >Ni2 >Zn2 。(2)萌发率为40%以上时,K 和Ca2 可以提高Ni2 、Zn2 和Co2 胁迫下油菜种子的萌发率,却进一步降低了Hg2 、Cd2 胁迫下种子的萌发;Mg2 可以提高Ni2 、Zn2 、Cd2 和Co2 胁迫下种子的萌发率,但对Hg2 毒害却没有缓解。(3)胚根伸长率达到60%以上时,K 和Mg2 增强了Ni2 、Hg2 、Cd2 和Co2 对胚根生长的抑制,而Ca2 则缓解了Zn2 、Ni2 和Co2 对胚根生长的抑制作用。研究结果对于重金属复合污染土壤中植物种子的萌发和定植具有理论和实践意义。     

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     

Inhibition of the Production of Phenolic Compounds in Brassica napus 2-Amino-oxyacetic Acid

Phenolic compounds precipitated with caffeine were more widelydistributed in the cytoplasm and nucleus in the outermost layerof the columella of germinating Brassica napus root than inthe other layers. Amino-oxyacetic acid, an inhibitor of thebiosynthesis of the C₆C₃ part of phenolic compounds, inhibitedthe biosynthesis of phenolics, and made dark deposits less numerousin all three of the outermost layers of the columella. The firstlayer was the most responsive, because symptoms of inhibitionwere already visible after 3 h. After 24 h the inhibition inthe second and third layers was proportional to the concentration.The first layer contained less deposits than the control, butmore than the second and third layers, suggesting that manyof these compounds in the first layer were present at the outset.Copyright1995, 1999 Academic Press Phenolics, 2-amino-oxyacetic acid, Brassica, germination, cytoplasm, nucleus     

Apoplastic pH and Ammonium Concentration in Leaves of Brassica napus L

A vacuum infiltration technique was developed that enabled the extraction of apoplastic solution with very little cytoplasmic contamination as evident from a malate dehydrogenase activity of less than 1% in the apoplastic solution relative to that in bulk leaf extracts. The volume of apoplastic water, a prerequisite for determination of the concentration of apoplastic solutes, was determined by vacuum infiltration of indigo carmine with subsequent analysis of the dilution of the dye in apoplastic extracts. Indigo carmine was neither transported across the cell membrane nor significantly adsorbed to the cell walls, ensuring reproducible (SE < 2%) and precise determination of apoplastic water. Analysis of leaves from four different positions on senescing Brassica napus plants showed a similar apoplastic pH of 5.8, while apoplastic NH4+ increased from 1.1 mM in lower leaves to 1.3 mM in upper leaves. Inhibition of glutamine synthetase in young B. napus plants resulted in increasing apoplastic pH from 6.0 to 6.8 and increasing apoplastic NH4+ concentration from 1.0 to 25.6 mM, followed by a marked increase in NH3 emission. Calculating NH3 compensation points for B. napus plants on the basis of measured apoplastic H+ and NH4+ concentrations gave values ranging from 4.3 to 5.9 nmol NH3 mol-1 air, consistent with an estimate of 5.3 [plus or minus] 3.6 nmol NH3 mol-1 air obtained by NH3 exchange experiments in growth chambers. A strong linear relationship was found between calculated NH3 compensation points and measured NH3 emission rates in glutamine synthetase-inhibited plants.     

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。     

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.     

Cytochemical Localization of Phenolic Compounds in Columella Cells of the Root Cap during Maturation of Seeds of Brassica napus L.

Abstract: Ultrastructural localization of phenolic compounds in the columella of the root cap of a canola embryo in Brassica napus L. during subsequent seed maturation stages was investigated. In bright green seeds the first signs of maturation and appearance of the first phenolic structures in the cell nuclei and cytoplasm in the area of the initial centre were found. Phenolics spread acropetally, gradually covering the whole columella in the dark green seeds. An increase in the number of phenolics occurred, together with an increase in the number of endoplasmic reticulum structures and in storage materials. In brown seeds, together with ultrastructural degradation and the end of production of storage substances, the number of phenolic deposits in the nucleus and cytoplasm decreases until their disappearance in black dormant seeds. Simultaneously, numerous phenolic deposits appear between the plasmalemma and the cell walls in the outer layer of the cap columella.     

Embryogenesis following cryopreservation in isolated microspores of rapeseed (Brassica napus L.)

A simple procedure is described for cryopreservation of isolated microspores of rapeseed in liquid nitrogen without loss of embryogenic capacity (i.e. embryogenes is can still be induced following freezing). Microspores frozen in Lichter's (1982) medium with 13% sucrose produced ca. 10% of the embryos yielded by an unfrozen control. Microspores frozen in Lichter's medium with 13% sucrose, and supplemented with 0.5 M glycerol and 0.5 M DMSO produced no embryos. Regeneration of embryos obtained from frozen microspores yielded 88% diploid and 12% haploid plants, while embryos from unfrozen controls produced 7% diploids and 93% haploids. The potential to increase the efficiency of the rapeseed haploidy system using cryopreservation is discussed in light of these results.     

Plasticity in Cell Division Patterns and Auxin Transport Dependency during in Vitro Embryogenesis in Brassica napus

In Arabidopsis thaliana, zygotic embryo divisions are highly regular, but it is not clear how embryo patterning is established in species or culture systems with irregular cell divisions. We investigated this using the Brassica napus microspore embryogenesis system, where the male gametophyte is reprogrammed in vitro to form haploid embryos in the absence of exogenous growth regulators. Microspore embryos are formed via two pathways: a zygotic-like pathway, characterized by initial suspensor formation followed by embryo proper formation from the distal cell of the suspensor, and a pathway characterized by initially unorganized embryos lacking a suspensor. Using embryo fate and auxin markers, we show that the zygotic-like pathway requires polar auxin transport for embryo proper specification from the suspensor, while the suspensorless pathway is polar auxin transport independent and marked by an initial auxin maximum, suggesting early embryo proper establishment in the absence of a basal suspensor. Polarity establishment in this suspensorless pathway was triggered and guided by rupture of the pollen exine. Irregular division patterns did not affect cell fate establishment in either pathway. These results confirm the importance of the suspensor and suspensor-driven auxin transport in patterning, but also uncover a mechanism where cell patterning is less regular and independent of auxin transport.     

Detection of Pollen Germination Inhibitors in Brassica oleracea Tissue Extracts

Petunia hybrida and Lilium lankongense pollens were germinatedon thin layer chromatography (TLC) plates following chromatographyof extracts from the self-, cross- and unpollinated stigmas,styles and ovaries and the seeds, leaves and pollen of threeinbred Brassica oleracea families. Zones of pollen germinationinhibition on the TLC plates showed that inhibitory compoundswere present in the tissue extracts. The R_f values and numberof these compounds varied with the tissue used, stigma tissuecontaining the largest amounts and the greatest number of inhibitors.In contrast, differences between the inbred lines tested wereslight and quantitative. Pollen from both P. hybrida and L.lankongense gave the same results; that from B. oleracea couldnot be used because of its poor germination. Brassica oleracea, Brussels sprout, kale, Lilium lankongense, Petunia hybrida, pollen germination, thin layer chromatography, germination inhibitors, phytoalexin, bioassay     

Interaction of Moisture Stress and Three Phenolic Compounds on Lettuce Seed Germination

No interactions between water stress and three phenolic acids(p-coumaric, caffeic and ferulic acids) on lettuce (Lactucasativa L. var. Grand Rapids) seed germination were found. Probitanalysis indicated that mechanisms of action of water stressand the phenolic inhibitors were similar. The relative effectivenessof the compounds was p-coumaric > ferulic > caffeic. Nointeraction was found between p-coumaric and ferulic acid, whereasantagonism was found between caffeic acid and each of the othertwo phenolic acids. Lactuca sativa L., lettuce, germination, phenolic compounds, moisture stress, allelopathy, seed     

Microspore Culture of Hybrids Between Brassica napus and B. campestris

Embryos and regenerated plants were produced by isolated microspore culture of inter-specific hybrids between Brassica napus and B. campestris. The NLN media with different sucrose concentrations and pH values were tested and a protocol for optimal microspore culture of B. carnpestris was identified. The reciprocal hybrids between UM921 (B. campestris) and 911186 (B. napus) had significant higher embryo yield than other cultured hybrids. Obvious improvement of embryo yield and quality was achieved when hybrid plants of reciprocal UM921 × 911186 were grown under 10 ℃/5 ℃ (day/night) condition. There was significant correlation between embryo yield and seeds per pod on hybrid plants but no correlation between pollen fertility and embryo yield was detected among cultured.hybrids. The majority of microspore-derived plants from the reciprocal B. napus × B. campestris hybrids are aneuploids and 22.8% of the plants observed originated from the microspores with parent′s chromosome numbers, almost all n = 19. The factors affecting the embryogenesis in microspore culture of interspecific hybrids and the possible applications of the technique are discussed.     

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     

壳聚糖包衣对油菜种子萌发及幼苗耐盐性影响

以不同浓度的壳聚糖对油菜种子进行包衣处理,考察其对油菜种子萌发及幼苗耐盐性的影响,并在不同盐浓度胁迫条件下对种子萌发时的发芽势、发芽率、生物量（鲜重、干重、根长、芽长）等指标进行测定,同时分析油菜幼苗叶绿素含量、可溶性蛋白及可溶性糖含量的变化。结果表明,一定浓度的壳聚糖包衣处理可提高油菜种子发芽率、发芽势、生物量、幼苗的耐盐指数、叶绿素含量、可溶性蛋白及可溶性糖的含量,其中浓度为0.25 g·L^-1壳聚糖包衣处理对油菜种子萌发的促进效果较好,而浓度为0.50 g·L^-1壳聚糖包衣处理对提高油菜幼苗耐盐性具有较好的促进作用。     

2个油菜CMS系统的酯酶和过氧化物酶同工酶分析

以甘蓝型油菜细胞质雄性不育系(CMS)Polima A及其保持系Polima B、陕2A及其保持系陕2B 4个材料初花期的叶片、叶柄以及花蕾组织为材料,采用聚丙烯酰胺垂直板凝胶电泳比较它们在酯酶(EST)和过氧化物酶(POD)同工酶酶谱的差异.结果表明,甘蓝型油菜不育系与其对应保持系的叶片、叶柄的EST同工酶谱均无明显差异,但两系花蕾的EST同工酶谱有一定的差异;Polima A与陕2A对应器官的EST同工酶谱均表现出明显差异.不育系与其对应保持系的叶片、叶柄的POD同工酶谱差异不明显,而两系花蕾的POD同工酶谱有明显的差异;两个不育系之间的POD同工酶谱明显不同.花蕾的EST和POD同工酶的条带数目明显多于相应材料的叶片和叶柄,且EST同工酶的条带数目明显多于相应的POD同工酶.因此,甘蓝型油菜CMS系统Polima A和陕2A有着不同的遗传背景.     

Germination stimulants of Phelipanche ramosa in the rhizosphere of Brassica napus are derived from the glucosinolate pathway

Phelipanche ramosa is a major parasitic weed of Brassica napus. The first step in a host-parasitic plant interaction is stimulation of parasite seed germination by compounds released from host roots. However, germination stimulants produced by B. napus have not been identified yet. In this study, we characterized the germination stimulants that accumulate in B. napus roots and are released into the rhizosphere. Eight glucosinolate-breakdown products were identified and quantified in B. napus roots by gas chromatography-mass spectrometry. Two (3-phenylpropanenitrile and 2-phenylethyl isothiocyanate [2-PEITC]) were identified in the B. napus rhizosphere. Among glucosinolate-breakdown products, P. ramosa germination was strongly and specifically triggered by isothiocyanates, indicating that 2-PEITC, in particular, plays a key role in the B. napus-P. ramosa interaction. Known strigolactones were not detected by ultraperformance liquid chromatography-tandem mass spectrometry, and seed of Phelipanche and Orobanche spp. that respond to strigolactones but not to isothiocyanates did not germinate in the rhizosphere of B. napus. Furthermore, both wild-type and strigolactone biosynthesis mutants of Arabidopsis thaliana Atccd7 and Atccd8 induced similar levels of P. ramosa seed germination, suggesting that compounds other than strigolactone function as germination stimulants for P. ramosa in other Brassicaceae spp. Our results open perspectives on the high adaptation potential of root-parasitic plants under host-driven selection pressures.     

Comparison of the genetic maps of Brassica napus and Brassica oleracea

 The genus Brassica consists of several hundreds of diploid and amphidiploid species. Most of the diploid species have eight, nine or ten pairs of chromosomes, known respectively as the B, C, and A genomes. Genetic maps were constructed for both B. napus and B. oleracea using mostly RFLP and RAPD markers. For the B. napus linkage map, 274 RFLPs, 66 RAPDs, and two STS loci were arranged in 19 major linkage groups and ten smaller unassigned segments, covering a genetic distance of 2125 cM. A genetic map of B. oleracea was constructed using the same set of RFLP probes and RAPD primers. The B. oleracea map consisted of 270 RFLPs, 31 RAPDs, one STS, three SCARs, one phenotypic and four isozyme marker loci, arranged into nine major linkage groups and four smaller unassigned segments, covering a genetic distance of 1606 cM. Comparison of the B. napus and B. oleracea linkage maps showed that eight out of nine B. oleracea linkage groups were conserved in the B. napus map. There were also regions in the B. oleracea map showing homoeologies with more than one linkage group in the B. napus map. These results provided molecular evidence for B. oleracea, or a closely related 2n=18 Brassica species, as the C-genome progenitor, and also reflected on the homoeology between the A and C genomes in B. napus. Received: 14 June 1996 / Accepted: 11 October 1996