Effects of aluminium on canola roots

There is little information on the effects of aluminium (Al) on canola (Brassica napus var. napus L.), which is a commercially important crop species in many parts of the world. In this report, we describe the effects of Al on roots of canola seedlings grown hydroponically in a nutrient solution at pH 4.5. The morphological and ultrastructural changes that accompanied these growth effects were examined. Additions to the nutrient solution of Al at concentrations below 40 μM stimulated root growth of canola seedlings, increasing both the size and number of central cap cells. The stimulation of root growth did not appear to be due to the alleviation of a proton toxicity at the root surface. At concentrations of Al above 60 μM, root growth was strongly inhibited, with cellular damage being observed primarily in peripheral root cap cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

The use of haploidy to develop plants that express several recessive traits using light-seeded canola (Brassica napus) as an example

Summary The use of haploidy to introgress recessive traits into Brassica napus canola is illustrated by describing the properties of doubled haploids obtained by microspore culture from crosses between a yellow-seeded rapeseed line (low erucic acid, high glucosinolate) and black-seeded canola. Of the 99 doubled haploid lines that were produced, 3 were yellow-seeded canola lines. This result was not significantly different than the predicted frequency of 1 in 64 for the homozygous recessive phenotype in a doubled haploid population segregating for six recessive genes. Thus, the study supports previous models of inheritance determined for yellow seededness and glucosinolate content in Brassica napus. Also, since the chances of obtaining a plant with the same characteristics in a F₂ population are 1 in 4,096, the underscore results the advantages of using haploidy to introgress recessive traits into Brassica napus canola.     

Accumulation of a Brazil nut albumin in seeds of transgenic canola results in enhanced levels of seed protein methionine

We have increased the methionine content of the seed proteins of a commercial winter variety of canola by expressing a chimeric gene encoding a methionine-rich seed protein from Brazil nut in the seeds of transgenic plants. Transgenic canola seeds accumulate the heterologous methionine-rich protein at levels which range from 1.7% to 4.0% of the total seed protein and contain up to 33% more methionine. The precursor of the methionine-rich protein is processed correctly in the seeds, resulting in the appearance of the mature protein in the 2S protein fraction. The 2S methionine-rich protein accumulates in the transgenic seeds at the same time in development as the canola 11S seed proteins and disappears rapidly upon germination of the seed. The increase in methionine in the canola seed proteins should increase the value of canola meal which is used in animal feed formulations.     

Hairy roots of Brassica napus: I. Applied glutamine overcomes the effect of phosphinothricin treatment

Summary Hairy roots of Brassica napus (rape cv. Giant) were produced by cocultivating leaf and cotyledon explants with Agrobacterium rhizogenes strain A4T. The hairy roots grew prolifically on solid and in liquid media. Incorporation of ammonium sulphate or phosphinothricin (PPT) into the media reduced growth. PPT treatment reduced glutamine synthetase (GS) activity and increased the ammonia content of the hairy roots. We have found that PPT treatment also induces a loss of glutamine from the roots and this may influence root growth. To test this we grew hairy roots in a liquid medium containing 10 mM glutamine. This glutamine treatment overcame the PPT induced suppression of growth but also significantly increased GS activity, reduced ammonia accumulation and increased the levels of glutamate and asparagine.     

Effects of sowing time and plant age on critical nitrogen concentrations in canola (Brassica napus L.)

Critical concentrations of NO₃-N in fresh petiole tissue and total N in the dried lamina were determined for the youngest mature leaf (YML) of field-grown canola. For dry matter yield of canola sown on 4 May, critical NO₃-N concentration in the YML petiole at the rosette stage (RS) was 1.46 mg/g fresh wt. At the flower-buds-visible stage (BV) it was 0.45 mg/g fresh wt. For seed yield the values were 1.72 and 0.53 mg/g fresh wt. Critical total N concentration in the YML lamina for dry matter yield were 69 mg/g dry wt. at RS and 57 at BV. For seed yield they were 71 and 59 mg/g dry wt. Critical NO₃-N concentrations in the YML petiole of canola sown on 30 May were reduced by 50%; critical total-N concentrations in the YML lamina were not reduced to the same extent. Despite the reductions in critical N concentrations in the YML, critical N fertilizer rates for vegetative growth and seed yield were unaffected by sowing date or plant growth stage.     

Stable expression of Phytase (phyA) in canola (Brassica napus) seeds: towards a commercial product

TheAspergillus niger gene encoding phytase(phyA) was expressed in canola (Brassicanapus). Phytase expression is controlled by the seed-specificcruciferin (CruA) promoter. Secretion of the enzyme was aimed for byincorporating the cruciferin signal peptide in the expression construct.Transgenic canola lines were generated by Agrobacteriummediated transformation using nptII as the selectable marker. Ninety-fiveindependent transgenic events were generated. Phytase expression in the T1seedsranged from 0 to 600 U/g seed. Single-copy lines were selected(based on segregation for kanamycin resistance, phytase expression and Southernanalyses) from originally multi-copy transgenic lines. Phytase was expressed inthese sub-lines up to 103 U/g. Expression levels were monitoredthrough an additional 3–4 generations (in the greenhouse and in thefield)and the accumulation of phytase appeared to be fairly stable. In the expressionrange studied, phytase expression was gene-dosage dependent.     

Intersubgenomic heterosis in seed yield potential observed in a new type of Brassica napus introgressed with partial Brassica rapa genome

This paper reports the observation on the intersubgenomic heterosis for seed yield among hybrids between natural Brassica napus (AⁿAⁿCⁿCⁿ) and a new type of B. napus with introgressions of genomic components of Brassica rapa (A^rA^r). This B. napus was selected from the progeny of B. napus × B. rapa and (B. napus × B. rapa) × B. rapa based on extensive phenotypic and cytological observation. Among the 129 studied partial intersubgenomic hybrids, which were obtained by randomly crossing 13 lines of the new type of B. napus in F₃ or BC₁F₃ to 27 cultivars of B. napus from different regions as tester lines, about 90% of combinations exceeded the yield of their respective tester lines, whereas about 75% and 25% of combinations surpassed two elite Chinese cultivars, respectively. This strong heterosis was further confirmed by reevaluating 2 out of the 129 combinations in a successive year and by surveying hybrids between 20 lines of the new type of B. napus in BC₁F₅ and its parental B. napus in two locations. Some DNA segments from B. rapa were identified with significant effects on seed yield and yield components of the new type of B. napus in BC₁F₅ and intersubgenomic hybrids in positive or negative direction. It seems that the genomic components introgressed from B. rapa contributed to improvement of seed yield of rapeseed.     

The glucosinolate-degrading enzyme myrosinase in Brassicaceae is encoded by a gene family

A full-length cDNA clone (MB3) and three partial clones (MA1, MB1 and MB2) which encode myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1) were isolated from a Sinapis alba (white mustard) cDNA library. Nucleotide sequence analysis of these clones revealed that they are encoded by a gene family. Southern blot analysis with gene-specific probes showed that the gene family consists of a least two subfamilies (MA and MB) each with several members both in S. alba and in Brassica napus (oilseed rape). In Arabidopsis thaliana (wall cress) only three myrosinase genes seem to be present. Northern blot analysis indicated that all the myrosinase mRNA species have the same size, approximately 1.95 kb.     

Levels of indole-3-acetic acid in intact and decapitated coleoptiles as determined by a specific and highly sensitive solid-phase enzyme immunoassay

A specific solid-phase enzyme immunoassay for the detection of as little as 3–4 pg of indole-3-acetic acid (IAA) is described. The assay involves minimal procedural efforts and requires only standard laboratory equipment. Up to 50 samples in triplicate, processed simultaneously, can be assayed and evaluated in 2.5 h. As little as 1 mg oat coleoptile tissue is sufficient for a quantitative IAA analysis and little or no extract purification is necessary. Using this assay, levels of IAA have been determined in coleoptiles of maize and oat. The distribution of IAA within single coleoptiles was quantitated and the production of IAA during the regeneration of the physiological tip in Avena coleoptiles was investigated. The changes in levels of IAA and other major phytohormones were quantitated during the growth of oat coleoptiles.Abbreviations ABA abscisic acid - BHT butylated hydroxytoluene - BSA bovine serum albumin - IAA indole-3-acetic acid - TBS Trishydroxymethylaminomethane buffered saline Part 21 in the series Use of Immunoassay in Plant Science     

A rapid visual method to identify transformed plants

Summary A rapid, efficient assay that is nondestructive and semiquantitative for identifying transgenic plants and progeny from Biolistic^? and protoplast transformations is described. Leaf sections of maize and wheat plants are placed on an indicator medium containing chlorophenol red and the selection agent. Changes in the color of the medium from red to yellow resulting from altered pH indicate transformed plants within 2-5 d. The method is particularly suited to use with phosphinothricin and could be used with other suitable selectable markers.     

A simple method to estimate the percentage of hybridity in canola (Brassica napus) F1 hybrids

We have developed an efficient PCR-based system that uses RAPD markers for the certification of F₁ hybrids of canola. These markers were selected by screening five parental lines used in three crosses X, Y and Z with 131, 131 and 322 primers respectively. Stable DNA fragments that were homozygous and specific to the male inbreds were used to certify F₁ hybrid populations. The hybrid production system was based on self-incompatibility (SI) alleles that prevent self-pollination of the female parent. The efficiency of two S-alleles was compared under both field and greenhouse conditions. The percentage of hybridity was estimated in different F₁ populations. We found a significant difference between the two alleles for their efficiency in controlling selfing; both alleles were stable under greenhouse conditions, one allele appeared less reliable under field conditions.     

Inhibition of CAT enzyme activity in Arabidopsis thaliana

Previously it was shown that transient chloramphenicol acetyltransferase (CAT) marker gene expression in Arabidopsis thaliana and Nicotiana tabacum resulted in significant differences in the accumulation of the CAT reaction products in radioactive CAT assays. Compared to Nicotiana tabacum, conversion of chloramphenicol to the acetylated products in Arabidopsis thaliana extracts was rather low. Here we report that the low CAT enzyme activity can be attributed in part to a heat sensitive CAT inhibitory effect in extracts of Arabidopsis thaliana. CAT enzyme activity in transgenic tobacco is inhibited by extracts from Arabidopsis. This inhibitory effect diminishes when Arabidopsis extracts were heat incubated. CAT activity in transgenic Arabidopsis lines was very low and was only detected in heat incubated extracts. Alternatively, enzyme-linked immunosorbent assays (ELISAs) can be used to detect the CAT protein in transgenic Arabidopsis.Abbreviations CAT chloramphenicol acetyltransferase - CAM chloramphenicol - ELISA enzyme linked immunosorbent assay     

Heterogeneity in the distribution of genetically modified and conventional oilseed rape within fields and seed lots

The implementation of co-existence in the commercialisation of GM crops requires GM and non-GM products to be segregated in production and supply. However, maintaining segregation in oilseed rape will be made difficult by the highly persistent nature of this species. An understanding of its population dynamics is needed to predict persistence and develop potential strategies for control, while to ensure segregation is being achieved, the production of GM oilseed rape must be accompanied by the monitoring of GM levels in crop or seed populations. Heterogeneity in the spatial distribution of oilseed rape has the potential to affect both control and monitoring and, although a universal phenomenon in arable weeds and harvested seed lots, spatial heterogeneity in oilseed rape populations remains to be demonstrated and quantified. Here we investigate the distribution of crop and volunteer populations in a commercial field before and during the cultivation of the first conventional oilseed rape (winter) crop since the cultivation of a GM glufosinate-tolerant oilseed rape crop (spring) three years previously. GM presence was detected by ELISA for the PAT protein in each of three morphologically distinguishable phenotypes: autumn germinating crop-type plants (3% GM), autumn-germinating 'regrowths' (72% GM) and spring germinating 'small-type' plants (17% GM). Statistical models (Poisson log-normal and binomial logit-normal) were used to describe the spatial distribution of these populations at multiple spatial scales in the field and of GM presence in the harvested seed lot. Heterogeneity was a consistent feature in the distribution of GM and conventional oilseed rape. Large trends across the field (50 x 400 m) and seed lot (4 x 1.5 x 1.5 m) were observed in addition to small-scale heterogeneity, less than 20 m in the field and 20 cm in the seed lot. The heterogeneity was greater for the 'regrowth' and 'small' phenotypes, which were likely to be volunteers and included most of the GM plants detected, than for the largely non-GM 'crop' phenotype. The implications of the volunteer heterogeneity for field management and GM-sampling are discussed.     

Field testing of transgenic rapeseed cv. Hero transformed with a yeast sn-2 acyltransferase results in increased oil content,erucic acid content and seed yield

A major goal of our research is to produce, by genetic manipulation, Brassica napus L. cultivars with higher levels of 22:1 in their seed oil than in present Canadian HEA cultivars developed through traditional breeding. Previously, we reported that transgenic expression of a mutated yeast sn-2 acyltransferase (SLC1-1) in industrial rapeseed cv. Hero resulted in increased seed oil content, increased proportions of erucic acid and increased average seed weight (Zou et al. 1997). Those results were reported only for plants grown in a controlled greenhouse setting. Here we report a summary of the results from two successive years of field trials with T₄ and T₅ generations of B. napus cv. Hero transformed with the SLC1-1 gene. These trials, conducted at Rosthern, Saskatchewan, in two very different growing seasons, show that the SLC1-1 transgenics clearly and consistently out-performed controls, with much increased oil and 22:1 contents, as well as yield, under varying field conditions.     

Methyl jasmonate elicits a differential antioxidant response in light- and dark-grown canola (Brassica napus) roots and shoots

Since jasmonates have been shown to mimic some of the plant'sresponses to stress, the effect of methyl jasmonate on antioxidant enzymes andcompounds was investigated in roots and shoots of light- and dark-grown canola(Brassica napus cv. Westar). The pattern of superoxidedismutase isoforms activity was also investigated. When enzyme activities werecalculated on a per gram of fresh weight basis, nearly all enzymes examinedshowed enhanced activity. However, when these activities were calculated basedon the amount of protein, methyl jasmonate induced an increase only insuperoxide dismutase activity in the roots of both light- and dark-grownseedlings. The ascorbate level was found to be higher in treated shoots,whereasthe glutathione level was found to be higher in treated roots. We conclude thatthe plant's antioxidant response to methyl jasmonate may be mainlydetermined by the type of tissue rather than by the light conditions. However,this last factor appeared to be involved in some antioxidant componentresponse,e.g. catalase activity and glutathione content.     

Loss of soybean trypsin inhibitor in callus as monitored by inhibition enzyme immunoassay

Summary A monoclonal antibody was produced against Kunitz soybean inhibitor (KSBTI) and used in an inhibition enzyme immunoassay (EIA). The inhibition EIA was as sensitive as competetive EIAs and was easily modified for other protein-antibody interactions. The KSBTI assay described detected KSBTI in complex mixtures from 100 μg/ml to 50 ng/ml and did not react with the Bowman-Birk trypsin inhibitor. The assay was used to examine levels of KSBTI inGlycine max hypocotyl-derived callus tissue. The developing hypocotyls contained 0.21 μg KSBTI per mg of fresh tissue. This level of KSBTI rapidly decreased when placed in culture and was undetectable 6 days later. The decrease in KSBTI correlated with the development of callus.     

Transformation of Brassica napus canola cultivars with Arabidopsis thaliana acetohydroxyacid synthase genes and analysis of herbicide resistance

Summary A survey of selected crop species and weeds was conducted to evaluate the inhibition of the enzyme acetohydroxyacid synthase (AHAS) and seedling growth in vitro by the sulfonylurea herbicides chlorsulfuron, DPX A7881, DPX L5300, DPX M6316 and the imidazolinone herbicides AC243,997, AC263,499, AC252,214. Particular attention was given to the Brassica species including canola cultivars and cruciferous weeds such as B. kaber (wild mustard) and Thlaspi arvense (stinkweed). Transgenic lines of B. napus cultivars Westar and Profit, which express the Arabidopsis thaliana wild-type AHAS gene or the mutant gene csr1-1 at levels similar to the resident AHAS genes, were generated and compared. The mutant gene was essential for resistance to the sulfonylurea chlorsulfuron but not to DPX A7881, which appeared to be tolerated by certain Brassica species. Cross-resistance to the imidazolinones did not occur. The level of resistance to chlorsulfuron in transgenic canola greatly exceeded the levels that were toxic to the Brassica species or cruciferous weeds. Direct selection of transgenic lines with chlorsulfuron sprayed at field levels under greenhouse conditions was achieved.