The Brassica napus extA promoter: a novel alternative promoter to CaMV 35S for directing transgene expression to young stem tissues and load bearing regions of transgenic apple trees (Malus pumila Mill.)

The Brassica napus extensin A gene is highly expressed in root tissue of oilseed rape. In an attempt to identify an effective root-specific promoter for biotechnological applications, we have examined the ability of the –940 extA promoter to drive expression of the gusA reporter gene in the vegetative tissues of apple (Malus pumila Mill cv. Greensleeves). Transgenic apple lines were produced by Agrobacterium tumefaciens-mediated transformation and GUS activity was analysed both quantitatively and qualitatively. The extA promoter was active in all tissues of young plants in all 15 clones examined. However Southern blot data suggested that only a proportion of the population contained the entire promoter and that others had suffered deletions of unknown length. This may have contributed to the variation seen in the quantitative and qualitative expression of GUS. Specific GUS activity was highest in the stems where it approached, and in some clones, exceeded that using the constitutive CaMV 35S promoter. Histochemical analysis confirmed that GUS was localised to tissues involved in structural support of the stem. Staining was particularly intense at nodal junctions where high tensile stress is exerted on the tissues. Maturing phloem tissues showed localisation of expression to the phloem parenchyma cells and phloem fibres. Transverse sections of the root revealed staining of primary procambial tissues including the young endodermis but no staining was seen in the cortex. Although the –940 extA promoter is clearly not root-specific in apple, it is likely to have useful biotechnological applications in tree species.     

Arrest of embryo development in Brassica napus mediated by modified Pseudomonas aeruginosa exotoxin A

Intracellularly expressed cytotoxins are useful tools both to study the action of plant regulatory sequences in transgenic plants and to modify plant phenotype. We have engineered a low mammalian toxicity derivative of Pseudomonas aeruginosa exotoxin A for intracellular expression in plant cells by fusing the ADP ribosylating domain of the exotoxin gene to plant regulatory sequences. The efficacy of exotoxin A on plant cells was demonstrated by transient expression of the modified exotoxin gene in tobacco protoplasts: the exotoxin gene inhibited the expression of a co-electroporated -glucuronidase gene. An exotoxin with an introduced frameshift mutation was also effective at inhibiting -glucuronidase expression in the transient assay; the activity of the frameshifted gene was presumably a result of frameshifting during translation or initiation of translation at a codon other than AUG. When fused to napin regulatory sequences, the exotoxin gene specifically arrested embryo development in the seeds of transgenic Brassica napus plants concomitant with the onset of napin expression. The napin/exotoxin chimeric gene did not have the same pattern of expression in tobacco as in B. napus; in addition to exhibiting an inhibition of seed development, the transgenic tobacco plants were male-sterile.     

The isolation and characterisation of the tapetum-specific Arabidopsis thaliana A9 gene

The Brassica napus cDNA clone A9 and the corresponding Arabidopsis thaliana gene have been sequenced. The B. napus cDNA and the A. thaliana gene encode proteins that are 73% identical and are predicted to be 10.3 kDa and 11.6 kDa in size respectively. Fusions of an RNase gene and the reporter gene -glucuronidase to the A. thaliana A9 promoter demonstrated that in tobacco the A9 promoter is active solely in tapetal cells. Promoter activity is first detectable in anthers prior to sporogenous cell meiosis and ceases during microspore premitotic interphase.The deduced A9 protein sequence has a pattern of cysteine residues that is present in a superfamily of seed plant proteins which contains seed storage proteins and several protease and -amylase inhibitors.     

The Brassica napus extA extensin gene negative regulatory region controls expression in response to mechanical stresses

In the present study the hypothesis that the ?433 to ?664 bp negative regulatory region (NRR) of the Brassica napus extA extensin promoter controls extA activation in response to externally applied weight loads was tested. When weight loads were applied to the nodal regions of transgenic tobacco plants containing extA promoter deletions fused to GUS, repression controlled by the NRR was overcome and GUS expression was induced only in the transgenics carrying the NRR. This proves that extensin expression in nodal regions is not developmentally controlled, but is induced in response to mechanical stresses, and is controlled by the NRR. It was also shown that the activation of the extA promoter during the development of lateral roots is a stress‐related response that is also under the control of the NRR but that the constitutive expression of extensin mRNA in the phloem of roots is not due to the mechanical forces the root experiences as it forces it way through the soil. Electrophoretic mobility shift assays using a 25 bp oligonucleotide have been used to show that an 8 bp consensus sequence from the NRR binds nuclear proteins. Wound‐induced signals regulating extensin gene expression are shown to travel bi‐directionally through the plant, from root to leaf and vice versa.     

The promoter of aBrassica napus polygalacturonase gene directs pollen expression ofβ-glucuronidase in transgenicBrassica plants

A 647-bp 5-flanking fragment obtained from genomic clone Sta 44G(2) belonging to a family of polygalacturonase genes expressed inBrassica napus pollen was fused to the-glucuronidase (GUS) marker gene. This fusion construct was introduced intoB. napus plants viaAgrobacterium tumefaciens transformation. Analysis of the transgenicB. napus plants revealed that this promoter fragment is sufficient to direct GUS expression specifically in the anther and that GUS activity increases in pollen during maturation.Abbreviation GUS -Glucuronidase     

Oviposition behaviour of Dasineura brassicae on a high- versus a low-quality Brassica host

The oviposition behaviour of the brassica pod midge, Dasineura brassicae Winn. (Diptera: Cecidomyiidae), on a preferred host, Brassica napus L. (summer oilseed rape) was compared to that on a nonpreferred, less suitable host for larval growth, Brassica juncea (L.) Coss & Czern (brown mustard). The experiments were done under field conditions with wild females.The number of landing females was significantly higher on B. napus than on B. juncea, indicating host differences in olfactory and/or visual stimuli. After landing, the behaviour differed in that females stayed longer and laid more egg batches on B. napus than on B. juncea plants. The probability that oviposition would occur after landing and the potentially adjustable egg batch size were similar on the high- and the low-quality host.A larger egg load on B. napus than on B. juncea can thus be attributed mainly to a higher landing rate and more repeated ovipositions occurring on B. napus.

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Comportement de ponte de Dasineura brassicae sur des Brassica de qualites élevée et basse

Résumé La comparaison porte sur le comportement de ponte de D. brassicae Winn. (Dept. Cecidomyiidae) sur Brassica napus L., hôte préféré, et sur B. juncea L., hôte non-préféré, qui convient moins au développement larvaire. Les expériences réalisées en champ ont porté sur des femelles sauvages.Le nombre de femelles qui atterrissent sur B. napus est significativement plus élevé que sur B. juncea, ce qui indique des différences entre les stimuli olfactif et visuel des plantes-hôtes. Aprés atterrisage, le comportement diffère en ce que les femelles restent plus longtemps et déposent plus de pontes sur B. napus que sur B. juncea. La probabilité pour que la ponte suive l'atterrissage et les tailles des ooplaques potentiellement ajustables sont semblables sur les hôtes de qualité élevée ou basse.Une charge supérieure en oeufs sur B. napus que sur B. juncea peut ainsi être principalement attribuée à un taux d'atterrissage plus élevé et à une plus grande fréquence de ponte sur B. napus.

     

Characterization of a gene highly expressed in the Brassica napus pistil that encodes a novel proline-rich protein

Pis 30, a gene highly expressed in Brassica napus pistils and encoding a novel proline-rich protein was isolated and characterized. Sequences homologous to the Brassica Pis 30 gene were found only in Arabidopsis thaliana. The Pis 30 gene encodes a mature protein of 8.4 kDa with no previously characterized protein domains and whose function remains unknown. PIS 30 contains especially high levels of Pro (33%), but also of Leu (14%), Phe (10%) and Ser (6%). Although it is a proline-rich protein, PIS 30 shows only limited similarity to previously characterized plant proline-rich proteins. When compared to the stigma-specific activity of the B. napus SLR1 gene promoter in pistils of transgenic Arabidopsis, an 808 bp Pis 30 promoter fragment directed -glucuronidase expression primarily in the ovary, as well as in the stigma.     

Alternaria incidence in some alloplasmic lines of Indian mustard (Brassica juncea (L.) Coss.)

Summary The cytoplasmic substitution lines of Brassica juncea (L.) Coss were evaluated for their field resistance to Alternaria blight (Alternaria brassicae). The euplasmic B. juncea cv. RLM 198 had a mesothetic reaction while alloplasmic B. juncea lines with cytoplasms of B. campestris, B. chinensis, and B. japonica were highly susceptible. B. nigra cytoplasm did not have any effect on the disease reaction of the B. juncea genome. However, the alloplasmic lines with the cytoplasm of B. napus and B. carinata revealed a comparatively higher degree of resistance. The study underlined the utility of cytoplasmic manipulations in modifying the phenotypic expression of nuclear genes.     

Antioxidant defense system in leaves of Indian mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>) and rape (<Emphasis Type="Italic">Brassica napus</Emphasis>) under cadmium stress

Plant species capable of hyper-accumulating heavy metals are of considerable interest for phytoremediation, and differ in their ability to accumulate metals from environment. Using two brassica species (Brassica juncea and Brassica napus), nutrient solution experiments were conducted to study variation in tolerance to cadmium (Cd) toxicity based on (1) lipid peroxidation and (2) changes in antioxidative defense system in leaves of both plants (i.e., superoxide dismutase (SOD EC 1.15.1.1), catalase (CAT EC 1.11.1.6), ascorbate peroxidase (APX EC 1.11.1.11), guaiacol peroxidase (GPX EC 1.11.1.7), glutathione reductase (GR EC 1.6.4.2), levels of phytochelatins (PCs), non-protein thiols (NP-SH), and glutathione. Plants were grown in nutrient solution under controlled environmental conditions, and subjected to increasing concentrations of Cd (0, 10, 25 and 50 μM) for 15 days. Results showed marked differences between both species. Brassica napus under Cd stress exhibited increased level of lipid peroxidation, as was evidenced by the increased malondialdehyde (MDA) content in leaves. However, in Brassica juncea treated plants, MDA content remained unchanged. In Brassica napus, with the exception of GPX, activity levels of some antioxidant enzymes involved in detoxification of reactive oxygen species (ROS), including SOD, CAT, GR, and APX, decreased drastically at high Cd concentrations. By contrast, in leaves of Brassica juncea treated plants, there was either only slight or no change in the activities of the antioxidative enzymes. Analysis of the profile of anionic isoenzymes of GPX revealed qualitative changes occurring during Cd exposure for both species. Moreover, levels of NP-SH and PCs, monitored as metal detoxifying responses, were much increased in leaves of Brassica juncea by increasing Cd supply, but did not change in Brassica napus. These results indicate that Brassica juncea plants possess the greater potential for Cd accumulation and tolerance than Brassica napus.     

Study of factors affecting the culture of Brassica napus L. and B. juncea Coss. mesophyll protoplasts

Various factors affecting the culture of Brassica napus and B. juncea mesophyll protoplasts were examined in order to develop suitable culture media for these species. The basic components (salts and vitamins) of culture media K3 and Kao best supported cell division and colony development in protoplast culture of both species. The addition of casamino acids to Kao's medium resulted in colony browning in B. napus genotypes. B. napus protoplasts grew well with glucose as the osmotic stabilizer, whereas B. juncea protoplasts responded better to sucrose. High NAA and low 2,4-D combinations were effective in stimulating colony growth. Colony development was rapid for a range of genotypes cultured with these recommendations in these media and plant regeneration was obtained from protoplast-derived calli in both species.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - BAP 6-Benzylaminopurine - MES 2(N-Morpholino)ethane sulfonic acid Contribution No. 931.     

Transient expression of β-glucuronidase in Arabidopsis thaliana leaves and roots and Brassica napus stems using a pneumatic particle gun

Successful transient expression of -glucuronidase (GUS) in Arabidopsis thaliana leaves and roots and Brassica napus stems was obtained after gene delivery with a pneumatic particle gun driven by compressed air. Effects of the pneumatic pressure used to accelerate the particles (accelerating pressure; 85 to 200 kg/cm²) and of preculture periods of plant tissues (0 to 6 days) on the efficiency of gene delivery were studied. In A. thaliana leaves, best results were obtained at 115 kg/cm² of accelerating pressure and 3 days of preculture. In A. thaliana roots, the optimum was at 200 kg/cm² of accelerating pressure and 3 days of preculture. These results indicate that both preculture period and accelerating pressure are vital factors that determine the efficiency of gene delivery by particle gun.     

The genetics of adult-plant blackleg (Leptosphaeria maculans) resistance from Brassica juncea in B. napus

The genetic control of adult-plant blackleg (Leptosphaeria maculans) resistance in a Brassica napus line (579NO48-109-DG-1589), designated R13 possessing Brassica juncea-like resistance (JR), was elucidated by the analysis of segregation ratios in F₂ and F₃ populations from a cross between R13 and the highly blackleg-susceptible B. napus cultivar Tower. The F₂ segregration ratios were bimodal, demonstrating that blackleg resistance in R13 was controlled by major genes. Analysis of the segregation ratios for 13 F₃ families indicated that blackleg resistance in these families was controlled by three nuclear genes, which exhibited a complex interaction. Randomly sampled plants of F₃ progeny all had the normal diploid somatic chromosome number for B. napus. The similarities between the action of the three genes found in this study with those controlling blackleg resistance in B. juncea is discussed.     

Virulence of Agrobacterium tumefaciens strains with Brassica napus and Brassica juncea

Summary Brassica napus and Brassica juncea were infected with a number of Agrobacterium tumefaciens strains. Tumourigenesis was very rapid and extremely efficient on B. juncea with all but one of the strains. Tumourigenesis on B. napus varied widely. It was very efficient with the nopaline strains, was reduced with the succinamopine strain A281 and was very weak with the octopine strains. The latter observation was confirmed with six different B. napus rapeseed cultivars. The selectivity was due to differences in the virulence of Ti plasmids with B. napus, rather than the tumourigenicity of the T-DNA or virulence of the chromosomal genes associated with the strains. An exception was strain LBA4404. The virulence of the octopine strains was increased by coinfection with more virulent disarmed strains and by induction with acetosyringone.     

Transforming petals into sepaloid organs in<Emphasis Type="Italic"> Arabidopsis</Emphasis> and oilseed rape: implementation of the hairpin RNA-mediated gene silencing technology in an organ-specific manner

Oilseed rape (Brassica napus L.) genotypes with no or small petals are thought to have advantages in photosynthetic activity. The flowers of field-grown oilseed rape form a bright-yellow canopy that reflects and absorbs nearly 60% of the photosynthetically active radiation (PAR), causing a severe yield penalty. Reducing the size of the petals and/or removing the reflecting colour will improve the transmission of PAR to the leaves and is expected to increase the crop productivity. In this study the hairpin RNA-mediated (hpRNA) gene silencing technology was implemented in Arabidopsis thaliana (L.) Heynh. and B. napus to silence B-type MADS-box floral organ identity genes in a second-whorl-specific manner. In Arabidopsis, silencing of B-type MADS-box genes was obtained by expressing B. napus APETALA3 (BAP3) or PISTILLATA (BPI) homologous self-complementary hpRNA constructs under control of the Arabidopsis A-type MADS-box gene APETALA1 (AP1) promoter. In B. napus, silencing of the BPI gene family was achieved by expressing a similar hpRNA construct as used in Arabidopsis under the control of a chimeric promoter consisting of a modified petal-specific Arabidopsis AP3 promoter fragment fused to the AP1 promoter. In this way, transgenic plants were generated producing male fertile flowers in which the petals were converted into sepals (Arabidopsis) or into sepaloid petals (B. napus). These novel flower phenotypes were stable and heritable in both species.Abbreviations PAR photosynthetically active radiation - ST-LS1 potato light-inducible tissue-specific ST-LS1 gene - GUS -glucuronidase     

油菜类型和品种对外来入侵杂草小子虉草的替代控制作用

外来入侵植物小子虉草（Phalaris minor Retz.）是世界公认的冬季农田恶性杂草,掌握农作物对其替代控制作用具有重要的研究价值。前期研究表明,油菜是替代控制小子虉草的优良农作物,然而,目前尚不清楚油菜类型与品种对其控制能力的影响。为此选取与小子虉草同域发生的不同类型（白菜型油菜、芥菜型油菜和甘蓝型油菜）油菜品种各3种,通过田间小区实验和室内化感作用测定,对比研究其对小子虉草的生长、繁殖、表型以及化感作用的影响。田间实验显示：竞争方式（种内或种间竞争）和油菜类型对小子虉草的地上生物量、种子数、株高、分枝数、叶面积和比叶面积存在极显著（P=0.0001）影响;而油菜品种对小子虉草的地上生物量（P=0.6064）、种子数（P=0.3577）、株高（P=0.4279）、分枝数（P=0.6357）、叶面积（P=0.8839）和比叶面积（P=0.3424）均无显著影响。3种类型油菜对小子虉草生长、繁殖以及表型的影响存在明显差异,其中芥菜型油菜对小子虉草的上述指标的影响最强,而白菜型油菜的影响最弱。室内生物测定显示,油菜对小子虉草具有化感抑制作用,当供试油菜叶片水提液浓度为0.1 g/mL时,小子虉草种子的萌发和幼苗的株高、根长、生物量均被显著抑制;研究也表明不同类型油菜对小子虉草的化感作用显著不同,同等条件下,芥菜型油菜对小子虉草的化感抑制作用最强。综上所述,油菜类型对外来入侵小子虉草的控制作用存在显著差异,其中芥菜型油菜对植物小子虉草的替代控制作用明显优于白菜型油菜和甘蓝型油菜,而其强的化感抑草特性或许是其强控草能力的原因之一。另外,本研究也为进一步利用油菜替代控制入侵植物小子虉草提供了参考。     

Assessment of microinjection for introducing DNA into uninuclear microspores of rapeseed

Approximately 2,000 embryogenic uninuclear microspores of rapeseed (Brassica napus) cv. Topas were intranuclearly injected with a chimaeric -glucuronidase (Escherichia coli Uid A) gene. Stable integration had not occurred among 55 plants that were regenerated. Coinjection of the dye Lucifer Yellow and detection of injected DNA by the polymerase chain reaction revealed high frequencies of transfer. However, the amount of DNA injected was less than 20 copies, which may have been insufficient for stable transformation of microspores.Abbreviations PCR polymerase chain reaction - GUS -glucuronidase