Age-Dependent Modifications and Further Localization of the CX 26-like Protein from Vicia faba L.

A strong age dependency together with alterations in the cellular distribution of CX 26 immunorelated protein(s) was found for differently developed leaves of Vicia faba L. With increasing age, an immunoreactive 40 kD band was observed in the soluble and microsomal fraction. In the cell wall protein preparation of young and fully differentiated leaves the 40 kD band was the minor constituent. A 33 kD polypeptide was dominantly localized in the microsomal fractions of all developmental stages and in SDS-extracts of total cell proteins of young leaves. A 21 kD protein together with a 16 kD polypeptide was associated with the cell wall fraction. The 21 kD protein, assumed to represent a plasmodesmatal constituent, was reduced with age. In SDS extracts, prepared from the different developmental stages of the leaves and of mesophyll protoplasts, the age-dependent appearance of the several immunostained bands was most obvious. A correlation of the 16, 33, and 40 kD bands to a turnover of the 21 kD protein is suggested. The reduced amount of the 21 kD protein with increasing age may be contemplated as an indication for a relative decrease of symplastic connections between cells of maturing leaves. This is in agreement with the results obtained by immunofluorescence studies using guard cell protoplasts. Here, observations pointed also to a reduction and final loss of CX 26-related protein at the protoplast surfaces.     

Multiple forms of phosphoenolpyruvate carboxylase in mesophyll, epidermal and guard cells of Vicia faba

The distribution of phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) in different leaf‐cell‐types and tissues of Vicia faba L. cv. 3‐fach Weiße was studied. The highest specific PEPCase activity was found in guard cell protoplasts (16.3 µmol mg⁻¹ protein h⁻¹) whereas for epidermal and mesophyll protoplasts remarkably lower specific activities were found (1.6 and 1.0 µmol mg⁻¹ protein h⁻¹, respectively). On chlorophyll and protoplast basis, a similar distribution of enzyme activity was observed. Compared with epidermal extracts, the specific PEPCase activity of mesophyll tissue was 17‐fold lower. Immunological studies with polyclonal antibodies to PEPCase indicated 3 immunoreactive proteins in epidermal tissue and guard cell protoplasts with molecular masses of 107 000, 110 000, and 112 000. Only the M_r 107 000 protein was found in extracts of mesophyll and epidermis protoplasts. Western immunoblots after native electrophoresis of epidermal and mesophyll proteins showed a significant difference in PEPCase mobility. It is assumed, that the immunostained proteins of M_r 110 000 and 112 000 represent isoforms or subunits of the PEPCase and that they are involved in stomatal movements.     

Immunological Evidence of Connexin-like Proteins in the Plasma Membrane of Vicia faba L.

Plasma membranes from three week old leaves of Vicia faba L. were enriched by aqueous two-phase partitioning to high purity. Plasma membrane proteins were immunoblotted with polyclonal, monospecific antibodies raised against mouse liver connexins (cx) 32 and 26. Immunostaining after treatments with cx 32 antibodies revealed the existence of a 29 kDa protein, clearly enriched in the plasma membrane fraction. An additional immunoreactive band of 20 kDa, possibly a degradation product of the 29 kDa protein, was found in the soluble fraction. When immunoblots were incubated with cx 26 antibodies, a 40 kDa band with a strong immunoresponse appeared, assumed to present the dimeric form of a 21 kDa, cx 26-like plant protein. The monomeric form could be only obtained when intact leaf material or mesophyll protoplasts from three week old plants were directly SDS-extracted. Furthermore, in young, one week old leaves, the monomer seems to exist in larger amounts, together with another crossreacting 35 kDa protein. The 29 kDa (cx 32-related) as well as the 40 kDa (cx 26-related) polypeptide is obviously located in the plasma membrane. The 40 kDa protein has to be considered as a new connexin-like plant protein.     

Secondary phenolic products in isolated guard cell,epidermal cell and mesophyll cell protoplasts from pea (Pisum sativum L.) leaves: Distribution and determination

Summary Guard cells and epidermal cells of the abaxial (lower) and adaxial (upper) epidermis ofPisum sativum L., mutant Argenteum, are the predominant sites of flavonoid accumulation within the leaf. This was demonstrated by the use of a new method of simultaneous isolation and separation of intact, highly-purified guard cell and epidermal cell protoplasts from both epidermal layers and of protoplasts from the mesophyll. Isolated guard and epidermal protoplasts retained flavonoid patterns of the parent epidermal tissue; quercetin 3-triglucoside and its p-coumaric acid ester as major constituents, kaempferol 3-triglucoside and its p-coumaric acid ester as minor compounds. Total flavonoid content in the lower epidermis was estimated to be ca. 80 fmol per guard cell protoplast and 500 fmol per epidermal cell protoplast. Protoplasts isolated from the upper epidermis had about 20–30% as much of these flavonoids. Mesophyll protoplasts retained only about 25 fmol total flavonoid per protoplast.By fluorescence microscopy, using the alkaline-induced yellow-green fluorescence characteristics of flavonols, we suggest that these flavonol glycosides are present in cell vacuoles. There was no indication for the presence of flavine-like compounds.Abbreviations uE adaxial (upper) epidermis - IE abaxial (lower) epidermis - GCP guard cell protoplasts - ECP epidermal cell protoplasts - MCP mesophyll cell protoplasts - PP protoplasts - HPLC high performance liquid chromatography - TLC thin layer chromatography - CC column chromatography - HOAc acetic acid     

Cellular Distribution of Calmodulin and Calmodulin-Binding Proteins in Vicia faba L.

The distribution of calmodulin (CaM) and CaM-binding proteins within Vicia faba was investigated. Both CaM and CaM-binding proteins were found to be differentially distributed among organs, tissues, and protoplast types. CaM levels, on a per protein basis, were found to be the highest in leaf epidermis, containing 3-fold higher levels of CaM than in total leaf. Similarly, guard cell and epidermal cell protoplasts were also found to have higher levels of CaM than mesophyll cell protoplasts. ¹²⁵I-CaM blot overlay assays were performed to qualitatively examine CaM-binding proteins in these protoplast types as well as in whole tissues and organs. CaM-binding proteins with M_r 52,000, 78,000, and 115,000 were common in all metabolically active plant parts. Unique CaM-binding protein bands were detected in guard cell protoplasts (M_r 39,000, 88,000), stems (M_r 45,000, 60,000, 64,000), and roots (M_r 62,000), suggesting the presence of specialized CaM-dependent processes in these cells and organs.     

Photophysiology of the Elongated Internode (ein) Mutant of Brassica rapa: ein Mutant Lacks a Detectable Phytochrome B-Like Polypeptide

Several phytochrome-controlled processes have been examined in etiolated and light-grown seedlings of a normal genotype and the elongated internode (ein/ein) mutant of rapid-cycling Brassica rapa. Although etiolated ein seedlings displayed normal sensitivity to prolonged far-red light with respect to inhibition of hypocotyl elongation, expansion of cotyledons, and synthesis of anthocyanin, they displayed reduced sensitivity to prolonged red light for all three of these deetiolation responses. In contrast to normal seedlings, light-grown ein seedlings did not show a growth promotion in response to end-of-day far-red irradiation. Additionally, whereas the first internode of light-grown normal seedlings showed a marked increase in elongation in response to reduced ratio of red to far-red light, ein seedlings showed only a small elongation response. When blots of protein extracts from etiolated and light-treated ein and normal seedlings were probed with monoclonal antibody to phytochrome A, an immunostaining band at about 120 kD was observed for both extracts. The immunostaining intensity of this band was substantially reduced for extracts of light-treated normal and ein seedlings. A mixture of three monoclonal antibodies directed against phytochrome B from Arabidopsis thaliana immunostained a band at about 120 kD for extracts of etiolated and light-treated normal seedlings. This band was undetectable in extracts of ein seedlings. We propose that ein is a photoreceptor mutant that is deficient in a light-stable phytochrome B-like species.     

水分胁迫下ABA由蚕豆根向地上部的运输及其在叶片组织中的分布

蚕豆根装载的３Ｈ－ＡＢＡ可经５．６ｃｍ／ｍｉｎ以上的速率向冠部运输。短时间内（５ｍｉｎ）根运来的ＡＢＡ主要分布在有大量气孔密布的下表皮,但长时间内（３ｈ）则主要分布在对内组织中。抑制蒸腾可降低ＡＢＡ向叶片中的运输积累。光镜放射自显影术显示,根运来的ＡＢＡ可有效地在表皮细胞及保卫细胞的质外体积累。３Ｈ－ＡＢＡ由根向地上部快速运输及其在作用部位的有效积累,说明水分胁迫下蚕豆根部可以通过ＡＢＡ信号的传递控制气孔的行为。     

Intergeneric protoplast fusion between Brassica carinata and Camelina sativa

Camelina sativa is a wild crucifer that is reported to be resistant to Alternaria blight. Polyethylene glycol mediated fusion was attempted between protoplasts from etiolated hypocotyls of Brassica carinata and mesophyll protoplasts of Camelina sativa. The mean frequency of heterokaryons was 6.8%. Three hybrid shoots were regenerated, each from a single fusionderived callus. These shoots failed to produce roots capable of withstanding transplantation. Confirmation of hybridity was obtained from the morphology of in vitro produced leaves, somatic chromosome number in leaf tips, and restriction fragment length polymorphism for a nuclear rDNA probe. Analysis for organelle constitution using RFLPs indicated that the hybrid contained chrloroplasts derived from the wild species and mitochondria from the cultivated Brassica species.Abbreviations 2,4-D 2,4-dichlorophenoxy-acetic acid - IAA Indole-3-acetic acid - NAA -Naphthaleneacetic acid - IBA Indole-3-butyric acid - GA₃ gibberellic acid - BAP 6-Benzylaminopurine - MS Murashige and Skoog (1962) basal medium     

TGD5 is required for normal morphogenesis of non-mesophyll plastids,but not mesophyll chloroplasts,in Arabidopsis

Stromules are dynamic membrane-bound tubular structures that emanate from plastids. Stromule formation is triggered in response to various stresses and during plant development, suggesting that stromules may have physiological and developmental roles in these processes. Despite the possible biological importance of stromules and their prevalence in green plants, their exact roles and formation mechanisms remain unclear. To explore these issues, we obtained Arabidopsis thaliana mutants with excess stromule formation in the leaf epidermis by microscopy-based screening. Here, we characterized one of these mutants, stromule biogenesis altered 1 (suba1). suba1 forms plastids with severely altered morphology in a variety of non-mesophyll tissues, such as leaf epidermis, hypocotyl epidermis, floral tissues, and pollen grains, but apparently normal leaf mesophyll chloroplasts. The suba1 mutation causes impaired chloroplast pigmentation and altered chloroplast ultrastructure in stomatal guard cells, as well as the aberrant accumulation of lipid droplets and their autophagic engulfment by the vacuole. The causal defective gene in suba1 is TRIGALACTOSYLDIACYLGLYCEROL5 (TGD5), which encodes a protein putatively involved in the endoplasmic reticulum (ER)-to-plastid lipid trafficking required for the ER pathway of thylakoid lipid assembly. These findings suggest that a non-mesophyll-specific mechanism maintains plastid morphology. The distinct mechanisms maintaining plastid morphology in mesophyll versus non-mesophyll plastids might be attributable, at least in part, to the differential contributions of the plastidial and ER pathways of lipid metabolism between mesophyll and non-mesophyll plastids.     

Malate Dehydrogenases in Guard Cells of Pisum sativum

Guard cell protoplasts of Pisum sativum show considerable NADP-dependent malate dehydrogenase (MDH) activity in darkness which can be enhanced severalfold by illumination or treatment with dithiothreitol (DTT). The question arose whether guard cells possess an NADP-MDH different from that present in the chloroplasts of the mesophyll (which is inactive in darkness or in the absence of DTT). MDH activities were determined in extracts of isolated protoplasts from mesophyll and epidermis, and in mechanically prepared epidermal pieces (with guard cells as the only living cells and no interference from proteases originating from the cell wall digesting enzymes). Guard cells possessed NAD-dependent MDHs of high activity and incomplete exclusion of NADP as a coenzyme. This NADP-dependent activity of the NAD-MDH(s) could not be stimulated by DTT or, inferentially, by light. The DTT- (and light-) dependent NADP-MDH represented 0.05% of the total protein of the guard cells and had a specific activity of 0.1 unit per milligram protein; both values are in the same range as the corresponding ones of the mesophyll cells. Agreement was also found in the extent of light activation, in subunit molecular weight, immunological cross-reactions, and in the behavior on an ion exchange column. The activity of the chloroplastic NADP-MDH in guard cells barely suffices to meet the malate requirement for stomatal opening in the light. It is therefore likely that NAD-MDHs residing in other compartments of the guard cells supplement the activity of the chloroplastic NADP-MDH particularly during stomatal opening in darkness.     

Nickel and zinc effects,accumulation and distribution in ruderal plants Lepidium ruderale and Capsella bursa-pastoris

Ruderal plants can grow in polluted areas, but little is known about heavy metal accumulation and distribution in them. Here Ni and Zn accumulation, distribution and effects were investigated in Lepidium ruderale and Capsella bursa-pastoris grown at 5–30 µM Ni(NO₃)₂ or 10–80 µM Zn(NO₃)₂. Metal contents were measured by flame atomic absorption spectrophotometry and tissue distribution of metals was studied histochemically. Ni was more toxic than Zn for both plants. When metal-induced growth-inhibiting effects were compared at various metal concentrations in solution, L. ruderale was more tolerant to Ni, whereas C. bursa-pastoris to Zn. However, when compared at similar Zn or Ni contents in roots, root growth of C. bursa-pastoris was more tolerant than that of L. ruderale. On the contrary, at similar Zn or Ni contents in shoots, shoot growth of L. ruderale was more tolerant. Both plants are excluders maintaining low metal levels in shoots. In roots, Ni located in protoplasts while Zn was also detected in cell walls. Metal accumulation in root apices resulted in growth inhibition. Ni accumulation in root cortex constrained metal translocation into central cylinder and then to shoots, where it located only in conductive tissues and epidermis, particularly in leaf trichomes of C. bursa-pastoris. Zn was translocated to shoots more actively and distributed in all shoot tissues, being accumulated in leaf vascular bundles and epidermis. To conclude, these patterns of Ni and Zn distribution are aimed at metal sequestration in roots and leaf epidermis, thus keeping mesophyll from metal penetration and pigment degradation.     

Differential tissue distribution of metabolites in Jacobaea vulgaris,Jacobaea aquatica and their crosses

Plants are attacked by many different herbivores. Some will consume whole leaves or roots, while others will attack specific types of tissue. Thus, insight into the metabolite profiles of different types of leaf tissues is necessary to understand plant resistance against herbivores. Jacobaea vulgaris, J. aquatica and three genotypes of their crossings were used to study the variation in metabolomic profiles between epidermis and mesophyll tissues. Extracts of epidermis and mesophyll tissues were obtained using carborundum abrasion (CA). Subsequently, ¹H nuclear magnetic resonance (NMR) spectroscopy and multivariate data analyses were applied to compare the metabolome profiles. Orthogonal partial least-squares-discriminant analysis (OPLS-DA) resulted in a clear separation of epidermis and mesophyll extracts. The epidermis contained significantly higher amounts of jacaranone and phenylpropanoids, specifically chlorogenic (5-O-CQA) and feruloyl quinic (FQA) acids compared to the mesophyll. In contrast, the mesophyll showed significantly higher concentrations of pyrrolizidine alkaloids (PAs), specifically jacobine and jaconine. The tissue specific distribution of these compounds was constant over all genotypes tested. Phenylpropanoids, 5-O-CQA and FQA, as well as PAs are known for their inhibitory effect on herbivores, especially against thrips. Thrips feeding commences with the penetration of the epidermis, followed by ingestion of sub-epidermal or mesophyll. Thrips thus may have to encounter phenylpropanoids in the epidermis as the first line of defence, before encountering the PAs as the ultimate defence in the mesophyll. The finding of tissue specific defense may have a major impact on studies of plant resistance. We cannot judge resistance using analyses of a whole roots, leafs or flowers. In such a whole-organism approach, the levels of potential defense compounds are far below the real ones encountered in tissues involved in the first line of defense. Instead, it is of great importance to study the defence compounds in the specific tissue to which the herbivore is confined.     

Immunochemical detection with rabbit polyclonal and mouse monoclonal antibodies of different pools of phytochrome from etiolated and green Avena shoots

While two monoclonal antibodies directed to phytochrome from etiolated oat (Avena sativa L.) shoots can precipitate up to about 30% of the photoreversible phytochrome isolated from green oat shoots, most precipitate little or none at all. These results are consistent with a report by J.G. Tokuhisa and P.H. Quail (1983, Plant Physiol. 72, Suppl., 85), according to which polyclonal rabbit antibodies directed to phytochrome from etiolated oat shoots bind only a small fraction of the phytochrome obtained from green oat shoots. The immunoprecipitation data reported here indicate that essentially all phytochrome isolated from green oat shoots is distinct from that obtained from etiolated oat shoots. The data indicate further that phytochrome from green oat shoots might itself be composed of two or more immunochemically distinct populations, each of which is distinct from phytochrome from etiolated shoots. Phytochrome isolated from light-grown, but norflurazon-bleached oat shoots is like that isolated from green oat shoots. When light-grown, green oat seedlings are kept in darkness for 48 h, however, much, if not all, of the phytochrome that reaccumulates is like that from etiolated oat shoots. Neither modification during purification from green oat shoots of phytochrome like that from etiolated oat shoots, nor non-specific interference by substances in extracts of green oat shoots, can explain the inability of antibodies to recognize phytochrome isolated from green oat shoots. Immunopurified polyclonal rabbit antibodies to phytochrome from etiolated pea (Pisum sativum L.). shoots precipitate more than 95% of the photoreversible phytochrome obtained from etiolated pea shoots, while no more than 75% of the pigment is precipitated when phytochrome is isolated from green pea shoots. These data indicate in preliminary fashion that an immunochemically unique pool of phytochrome might also be present in extracts of green pea shoots.Abbreviation ELISA enzyme-linked immunosorbent assay - mU milliunit - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome     

Tissue Distribution of beta-Cyanoalanine Synthase in Leaves

β-Cyanoalanine synthase, which catalyzes the reaction between cysteine and HCN to form β-cyanoalanine and H₂S, was assayed in leaf tissues from cyanogenic (Sorghum bicolor × Sorghum sudanense [sorghum]) and noncyanogenic (Pisum sativum [pea], Zea mays [maize], and Allium porrum [leek]) plants. The activity in whole leaf extracts ranged from 33 nanomoles per gram fresh weight per minute in leeks, to 1940 nanomoles per gram fresh weight per minute in sorghum. The specific activities of β-cyanoalanine synthase in epidermal protoplasts from maize and sorghum and in epidermal tissues from peas were in each case greater than the corresponding values for mesophyll protoplasts or tissues, or for strands of bundle sheath cells.

The tissue distributions for this enzyme were determined for pea, leek, and sorghum: the mesophyll protoplasts and tissues in these three plants contained 65% to 78% of the enzyme, while epidermal protoplasts and tissues contained 10% to 35% of the total leaf activity. In sorghum, the bundle sheath strands contained 13% of the leaf activity. The presence of β-cyanoalanine synthase in all tissues and species studied suggests a fundamental role for this enzyme in plant metabolism.

     

Guard Cell Chloroplasts Are Essential for Blue Light-Dependent Stomatal Opening in Arabidopsis

Blue light (BL) induces stomatal opening through the activation of H⁺-ATPases with subsequent ion accumulation in guard cells. In most plant species, red light (RL) enhances BL-dependent stomatal opening. This RL effect is attributable to the chloroplasts of guard cell, the only cells in the epidermis possessing this organelle. To clarify the role of chloroplasts in stomatal regulation, we investigated the effects of RL on BL-dependent stomatal opening in isolated epidermis, guard cell protoplasts, and intact leaves of Arabidopsis thaliana. In isolated epidermal tissues and intact leaves, weak BL superimposed on RL enhanced stomatal opening while BL alone was less effective. In guard cell protoplasts, RL enhanced BL-dependent H⁺-pumping and DCMU, a photosynthetic electron transport inhibitor, eliminated this effect. RL enhanced phosphorylation levels of the H⁺-ATPase in response to BL, but this RL effect was not suppressed by DCMU. Furthermore, DCMU inhibited both RL-induced and BL-dependent stomatal opening in intact leaves. The photosynthetic rate in leaves correlated positively with BL-dependent stomatal opening in the presence of DCMU. We conclude that guard cell chloroplasts provide ATP and/or reducing equivalents that fuel BL-dependent stomatal opening, and that they indirectly monitor photosynthetic CO₂ fixation in mesophyll chloroplasts by absorbing PAR in the epidermis.     

Cell-cell communication in the leaves of Commelina cyanea and other plants

Abstract. The fluorescent probes 6–carboxyfluorescein and lucifer yellow CH which do not pass the plasmalemma have been used to examine cell-to-cell communication in the leaf of Commelina cyanea. Dye movement from cell-to-cell occurs in epidermal, spongy and palisade mesophyll, and vascular cells. Dye movement between these tissues was also found. Hence, the epidermis, spongy and palisade mesophyll cells, and vascular tissue are all linked in a continuous symplast. However, dye injected into the epidermal cells rarely moves into guard cells, indicating that these cells are relatively isolated from the surrounding cells. In the same way, guard cells in Vicia faba and the C₄ grass Anthephora pubescens also appeared to be isolated from epidermal cells. Thus guard cell isolation from cell-to-cell communication appears to be a common phenomenon. Hence, the ion fluxes required for guard cell function must occur via the apoplast.     

Plating of isolated tobacco mesophyll protoplasts on agar medium

Summary A technique was developed to derive cell and plant clones from isolated mesophyll protoplasts of tobacco. The protoplasts, plated on a fully defined agar medium, divided and grew actively forming visible colonies after one month of culture. Efficiency of colony formation depended on cell density and light condition during incubation. Under standard conditions, 60% of plated protoplasts formed colonies. Upon transfer onto suitable media, these colonies differentiated shoots and roots, and eventually regenerated whole plants. Advantages of mesophyll protoplasts as the source of clones as well as implication of the plating technique for genetical studies are discussed.     

Water permeability differs between growing and non-growing barley leaf tissues

A pressure probe technique and an osmotic swelling assay were used to compare water transport properties between growing and non-growing tissues of leaf three of barley. The epidermis was analysed in planta by pressure probe, whereas (predominantly) mesophyll protoplasts were analysed by osmotic swelling. Hydraulic conductivity (Lp) and, by implication, water permeability (Pf) of epidermal cells was 31% higher in the leaf elongation zone (Lp=0.5+/-0.2 microm s-1 MPa-1; Pf=65+/-25 microm s-1; means+/-SD of n=17 cells) than in the, non-growing, emerged leaf zone (Lp=0.4+/-0.1 microm s-1 MPa-1; Pf=50+/-15 microm s-1; n=24; P<0.05). Similarly, water permeability of mesophyll protoplasts was by 55% higher in the elongation compared with emerged leaf zone (Pf=13+/-1 microm s-1 compared with 8+/-1 microm s-1; n=57 and 36 protoplasts, respectively; P<0.01). Within the leaf elongation zone, a small population of larger-sized protoplasts could be distinguished. These protoplasts, which originated most likely from parenchymateous bundle sheath or midrib parenchyma cells, had a three-fold higher water permeability (P<0.001) as mesophyll protoplasts. The effect on Lp and Pf of known aquaporin inhibitors was tested with the pressure probe (Au+, Ag+, Hg2+, phloretin) and the osmotic swelling assay (phloretin). Only phloretin, when applied to protoplasts in the swelling assay caused an average decrease in Pf, but the effect varied between isolations. Technical approaches and cell-type and growth-specific differences in water transport properties are discussed.     

Analysis of plants regenerated from protoplast fusions between Brassica napus and Eruca sativa

Summary Protoplasts from etiolated hypocotyls of Brassica napus stained with carboxyfluorescein were fused with mesophyll protoplasts from Eruca sativa. Hybrid cells could be identified under the light microscope by (1) fully developed chloroplasts derived from E. sativa and (2) the cytoplasmic strands of the B. napus hypocotyl protoplasts, or (3) by the presence of both red and green fluorescence when investigated under UV light. The heterokaryons were selected using either a micro-manipulator or a flow sorter. On average, 5.4% of the calli obtained after selection differentiated into shoots. Regenerated shoots were subjected to isozyme analysis for verification of their hybrid character. Of the 23 hybrids successfully transferred to the greenhouse, 11 were asymmetric according to isozyme analysis. The nuclear DNA content of the hybrids was determined by flow cytometry, which gives an estimate of chromosome number. Most of the hybrids had a DNA content, and thus a chromosome number, that deviated from the expected sum of the parents. Almost all of the hybrids had some degree of fertility and produced seeds. Seed set, expressed as seeds per pollinated flower, was on average 7% of that of B. napus in the case of self-pollination and 26% of that of B. napus when backcrossed to B. napus. The chloroplast genotype was investigated in 13 hybrids. Of these, 11 had chloroplasts derived from B. napus, while only 2 had chloroplasts of E. sativa origin.