Transgenic Oilseed Rape Plants Research Articles

BnaA03.MKK5-BnaA06.MPK3/BnaC03.MPK3 Module Positively Contributes to Sclerotinia sclerotiorum Resistance in Brassica napus.

Brassica napus (oilseed rape) is one of the most important oil crops worldwide, but its growth is seriously threatened by Sclerotinia sclerotiorum. The mechanism of oilseed rape response to this pathogen has rarely been studied. Here, it was identified that BnaA03.MKK5 whose expression was induced by S. sclerotiorum infection was involved in plant immunity. BnaA03.MKK5 overexpression lines exhibited decreased disease symptoms compared to wild-type plants, accompanied by the increased expression of camalexin-biosynthesis-related genes, including BnPAD3 and BnCYP71A13. In addition, two copies of BnMPK3 (BnA06.MPK3 and BnC03.MPK3) were induced by Sclerotinia incubation, and BnaA03.MKK5 interacted with BnaA06.MPK3/BnaC03.MPK3 in yeast. These interactions were confirmed using in vivo co-immunoprecipitation assays. In vitro phosphorylation assays showed that BnaA06.MPK3 and BnaC03.MPK3 were the direct phosphorylation substrates of BnaA03.MKK5. The transgenic oilseed rape plants including BnaA06.MPK3 and BnaC03.MPK3 overexpression lines and BnMPK3 gene editing lines mediated by CRISPR/Cas9 were generated; the results of the genetic transformation of BnaA06.MPK3/BnaC03.MPK3 indicate that BnMPK3 also has a positive role in Sclerotinia resistance. This study provides information about the potential mechanism of B. napus defense against S. Sclerotiorum mediated by a detailed BnaA03.MKK5-BnaA06.MPK3/BnaC03.MPK3 module.

Cre-mediated marker gene removal for production of biosafe commercial oilseed rape

The Cre/loxP-based self-excision represents a promising strategy for removal of hazardous transgene(s) from genomes of targeted crops prior to their introduction into environment. Here, we applied the Cre/loxP self-excision strategy in which the cre recombinase gene is driven by the embryo-specific CRUC promoter from Arabidopsis thaliana. Besides, the Cre/loxP cassette, the T-DNA also consisted of the β-glucuronidase gene, controlled by the double dCaMV 35S promoter and the selectable neomycin phosphotransferase gene; the latter was aimed to be removed from the transgenic genome. The Cre/loxP self-excision cassette was introduced into genomes of four commercial oilseed rape cultivars Haydn, Heros, Hunter and Topas (Brassica napus L.) via Agrobacterium tumefaciens. Transgenic T0 plants were regenerated from all cultivars. Their detailed molecular analyses revealed premature activation of the CRUC promoter that resulted in partial excision of the nptII gene. Progenies of four self-pollinated T0 lines were further analysed, and the data confirmed complete excision event in 5 out of 105 (4.8%) of T1 transgenic oilseed rape plants. The excision efficiency does not seem to depend on the target cultivar. However, the poor transformation efficiency of rapeseed and the limited specificity of the CRUC promoter are clearly the bottleneck of this approach, and the feasibility of (tissue-specific) self-excision of selectable marker gene from genomes of each commercial rapeseed variety adds to their perspective to cope the increasing negative impacts of climate changes.

Overexpression of an nsLTPs-like antimicrobial protein gene (LJAMP2) from motherwort (Leonurus japonicus) enhances resistance to Sclerotinia sclerotiorum in oilseed rape (Brassica napus)

Sclerotinia stem rot caused by Sclerotinia sclerotiorum is one of the most important diseases of oilseed rape worldwide and leads to considerable yield losses. In this study, a non-specific lipid transfer protein-like antimicrobial protein gene (LJAMP2) from motherwort (Leonurus japonicus) was introduced into oilseed rape (Zhongyou 821) by Agrobacterium-mediated transformation. In vitro experiments revealed that the mycelial growth of S. sclerotiorum was significantly inhibited when supplied with crude leaf extracts from transgenic oilseed rape plants overexpressing LJAMP2. Furthermore, in vivo studies showed that transgenic LJAMP2 plants had enhanced resistance to S. sclerotiorum. Semi-quantitative RT-PCR analysis showed that the LJAMP2 gene was transcribed in all transformed plants. In addition, we also found that overexpression of LJAMP2 in transgenic plants caused constitutive activation of the defense-related gene PR-1 and an increase of H2O2 production, but did not enhance PDF1.2 expression. Our results suggest that constitutive expression of the LJAMP2 gene from motherwort seeds might be exploited to improve the resistance of oilseed rape against S. sclerotiorum.

유채 조직배양 및 형질전환 연구동향

Oilseed rape (Brassica napus L.) is an important crop due to its high oil content in the seed. Recently, the demand for the improvement of crop for biodisel energy source is increased as oil prices in the world has increased dramatically. Until now, oilseed rape breeding was carried out by cross-hybridization between different varieties and related germplasms. However, like as many other crops, the application of tissue culture and gene transformation systems has been introduced into oilseed rape breeding program including the development of transgenic canola plants. In this study, we reviewed a history of tissue culture and genetic transformation research in oilseed rape plants and indicated some important aspects for the production of transgenic oilseed rape plants.

Bt-toxin uptake by the non-target herbivore,Myzus persicae(Hemiptera: Aphididae), feeding on transgenic oilseed rape in laboratory conditions

The potential non-target effects of genetically modified crops are some of the more debated topics within applied biotechnologies in agriculture and environmental risk assessment. The objective of the present research was to study the potential Bt-toxin uptake by the non-target herbivore Myzus persicae Sulzer (Hemiptera: Aphididae) feeding on transgenic oilseed rape plants (Brassica napus cv. 'Westar' lines GT 2-4) expressing the Cry1Ac endotoxin. A specific aim was to replicate our previous experiment in controlled laboratory conditions to avoid or minimize the risk of contamination leading to potential false positive results. The toxin levels in vernalized (V) and not-vernalized (not-V) transgenic oilseed rape plants was also monitored to better clarify the role of physiological processes on Bt-toxin expression. Cry1Ac expression in not-V plants (mean concentration±SE=167.8±5.7 μg kg-1 FW) showed a pattern of large variability, in comparison with V plants whose expression (mean concentration±SE=227.7±1.9 μg kg-1 FW) was significantly more stable. Cry1Ac toxin was detected in three aphid samples reared on V plants with a mean toxin concentration±SE of 4.8±0.6 μg Kg-1 FW and in three out of six samples of aphids reared on not-V plants (mean toxin concentration±SE=7.1±1.2 μg kg-1 FW). The mean Bt-toxin concentration of all the positive aphid samples was 5.9±1.0 μg kg-1 FW. Our results confirmed the findings of our previous experiment and highlighted the potential for Cry1Ac toxin uptake by aphids feeding on transgenic oilseed rape plants.

Expression of green fluorescent protein in pollen of oilseed rape (Brassica napus L.) and its utility for assessing pollen movement in the field

Transgene movement via pollen is an important component of gene flow from transgenic plants. Here, we present proof-of-concept studies that demonstrate the monitoring of short distant movement of pollen expressing a genetically encoded fluorescent tag in oilseed rape (Brassica napus L. cv. Westar). Transgenic oilseed rape plants were produced using Agrobacterium-mediated transformation method with the pBINDC1 construct containing a green fluorescent protein (GFP) variant, mGFP5-ER, under the control of the pollen-specific LAT59 promoter from tomato. Transgenic pollen was differentiated from non-transgenic pollen in vivo by a unique spectral signature, and was shown to be an effective tool to monitor pollen movement in the greenhouse and field. GFP-tagged pollen also served as a practical marker to determine the zygosity of plants. In a greenhouse pollen flow study, more pollen was captured at closer distances from the source plant plot with consistent wind generated by a fan. Under field conditions, GFP transgenic pollen grains were detected up to a distance of 15 m, the farthest distance from source plants assayed. GFP-tagged pollen was easily distinguishable from non-transgenic pollen using an epifluorescence microscope.

Monitoring the escape of transgenic oilseed rape around Japanese ports and roadsides

An investigation was carried out to monitor the escape and spread of oilseed rape (Brassica napus) transgenic plants and the introgression of transgenes to its closely related feral species in Japan. We screened a total of about 7500 feral B. napus, 300 B. rapa, and 5800 B. juncea seedlings from maternal plants in 143 locations at several ports, roadsides, and riverbanks. The presence of glufosinate-resistance or glyphosate-resistance transgenes in these seedlings was confirmed by means of herbicide treatments and also immunochemical and DNA analyses. B. napus plants with herbicide-resistant transgenic seeds were found at five of six major ports and along two of four sampled roadsides in the Kanto District. Transgenic oilseed rape plants have not been commercially cultivated in Japan, suggesting that the transgenes would probably have come from imported transgenic seeds that were spilled during transportation to oilseed processing facilities. No transgenes were detected in seeds collected from B. napus plants growing along riverbanks in the Kanto District or in seeds from closely related species (B. rapa and B. juncea). To our knowledge, this is the first published example of feral, transgenic populations occurring in a nation where the transgenic crop has not been cultivated commercially.

Effects of the cysteine protease inhibitor oryzacystatin (OC-I) on different aphids and reduced performance of Myzus persicae on OC-I expressing transgenic oilseed rape

When administered in artificial diets, the cysteine protease inhibitor (PI) oryzacystatin I (OC-I) induced moderate but significant growth inhibition on the pea aphid ( Acyrthosiphon pisum Harris), the cotton/melon aphid ( Aphis Gossypii Glover) and the peach potato aphid ( Myzus persicae Sulzer). In transgenic oilseed rape plants ( Brassica napus L. cv. Drakkar) expressing OC-I under the control of the CaMV-35S RNA promoter, oryzacystatin was detected in both leaves and phloem sap. Transgenic plants from three independent homozygous lines were used to test the effect of in planta-expression of OC-I on M. persicae. Mean adult weight and fecundity were significantly reduced, and aphid biomass produced in 2 weeks was decreased by 25–40% for aphids fed transgenic plants, when compared to those fed control plants. The effects of OC-I on M. persicae were correlated with the decrease of a major cathepsin L/H-type cysteine protease activity, detected in whole insect extracts. Immuno-histological analysis showed OC-I labeling along the gut epithelium, but also its association with aphid bacteriocytes and oenocytes. These results suggest that OC-I affects M. persicae through digestive tract targets, but also by reaching the haemolymph, thereby inhibiting extra-digestive proteolytic activities and interacting with functions related to aphid reproduction. Overall, it appears that PIs can display deleterious effects against phloem-feeding insects in addition to their activity on leaf-feeding insects.

Local and systemic resistance to fungal pathogens triggered by an AVR9-mediated hypersensitive response in tomato and oilseed rape carrying the Cf-9 resistance gene

Tomato and transgenic oilseed rape plants expressing the Cf-9 resistance gene develop a hypersensitive response (HR) after injection of the corresponding Avr9 gene product. It was investigated whether induction of a HR conferred resistance to different fungal pathogens in tomato and oilseed rape. Induction of an AVR9 mediated HR at the pathogen infection site delayed the development of the biotrophs Oidium lycopersicum in tomato and Erysiphe polygoni in oilseed rape, but enhanced the development of the necrotrophs Botrytis cinerea and Alternaria solani in tomato and Sclerotinia sclerotiorum in oilseed rape. Interestingly, delayed fungal disease development was observed in plant tissues surrounding the HR lesion regardless of whether a necrotrophic or biotrophic pathogen was used. In tomato, AVR9 injection induced systemic expression of PR1, PR2 and PR3 defence genes but did not induce systemic resistance to O. lycopersicum, B. cinerea or A. solani. In oilseed rape, AVR9 injection temporarily induced systemic resistance to Leptosphaeria maculans and E. polygoni, but did not induce detectable systemic expression of PR1, PR2 or Cxc750. These results give new insights into the potential uses of an induced HR to engineer disease resistance.

Functional expression of Cf9 and Avr9 genes in Brassica napus induces enhanced resistance to Leptosphaeria maculans.

The tomato Cf9 resistance gene induces an Avr9-dependent hypersensitive response (HR) in tomato and transgenic Solanaceae spp. We studied whether the Cf9 gene product responded functionally to the corresponding Avr9 gene product when introduced in a heterologous plant species. We successfully expressed the Cf9 gene under control of its own promoter and the Avr9 or Avr9R8K genes under control of the p35S1 promoter in transgenic oilseed rape. We demonstrated that the transgenic oilseed rape plants produced the Avr9 elicitor with the same specific necrosis-inducing activity as reported for Cladosporium fulvum. An Avr9-dependent HR was induced in Cf9 oilseed rape upon injection of intercellular fluid containing Avr9. We showed Avr9-specific induction of PR1, PR2, and Cxc750 defense genes in oilseed rape expressing CJ9. Cf9 x Avr9 oilseed rape did not result in seedling death of the F1 progeny, independent of the promoters used to express the genes. The F1 (Cf9 x Avr9) plants, however, were quantitatively more resistant to Leptosphaeria maculans. Phytopathological analyses revealed that disease development of L. maculans was delayed when the pathogen was applied on an Avr9-mediated HR site. We demonstrate that the CJ9 and Avr9 gene can be functionally expressed in a heterologous plant species and that the two components confer an increase in disease resistance.

Physiological adaptation explains the insensitivity of Baris coerulescens to transgenic oilseed rape expressing oryzacystatin I

Larvae of Baris coerulescens Scop. (Coleoptera: Curculionidæ) exhibit a complex array of gut proteinase activities comprising cysteine and serine proteinases. The major cysteine proteinase activity, showing an optimum at pH 6.0, corresponds to at least 4 different proteinases. On the contrary, the minor serine proteinase activity, with an optimum at pH 9.0, seems to be due essentially to a single proteinase. The cysteine proteinase inhibitor oryzacystatin I (OC-I) inhibits completely the cysteine proteinase activity in vitro. However, larval growth and survival were not significantly different on control and transgenic oilseed rape plants expressing high levels of active OC-I. In larvae grown on transgenic plants, cysteine proteinase activity was dramatically decreased, whereas serine proteinase activity was increased by more than 2-fold, when compared to larvae raised on control plants. For both activities, no new proteinase was detected in insects fed plants expressing OC-I. These results suggest that partial compensation of the inhibition of cysteine proteinase activity by the increase in serine proteinase activity allowed the larvae to overcome the effects of OC-I consumption. This case illustrates problems that could arise when trying to achieve high levels of protection for plants against Coleopteran pests possessing a complex digestive proteinase pool.

Occasional loss of expression of phosphinothricin tolerance in sexual offspring of transgenic oilseed rape (Brassica napus L.)

The commercial and economic value of genetically modified crops is determined by a predictable, consistent and stable transmission and expression of the transgenes in successive generations. No gene inactivation is expected after selfings or crosses with non-transformed plants of homozygous transgenic oilseed rape plants if the expression of the transgene in homozygous or hemizygous nature in such plants is stable. The segregation ratios of phosphinothricin (PPT) tolerance in successive generations of selfings and mutual crosses of a few independent transgenic PPT-tolerant oilseed rape plants indicated a dominant, monogenic inheritance. In within-variety and between-variety crosses no transgene inactivation was observed. However, after selfings and backcrosses with non-transgenic oilseed rape infrequent loss of the expression of the PPT tolerance transgene was observed independent from its homozygous or hemizygous nature. Molecular analysis of PPT-susceptible plants showed that the loss of expression was due to gene inactivation and not to the absence of the transgene. Methylation and co-suppression are mechanisms that might cause reduced or even loss of expression of the transgene in later generations. The implications of this observation for seed multiplication of varieties and breeding activities with transgenic oilseed rape are discussed.

Degradation of oxalic acid by transgenic oilseed rape plants expressing oxalate oxidase

Degradation of oxalic acid by transgenic oilseed rape plants expressing oxalate oxidase

Transformation of Brassica napus L. (oilseed rape) using Agrobacterium tumefaciens and Agrobacterium rhizogenes — A comparison

We have produced transgenic oilseed rape plants by two different transformation strategies. The first method utilised an A. rhizogenes binary system comprising of p Ri1855 and p Bin19 plasmids, to induce hairy root proliferation at the cotyledonary nodes. Clonal hairy root explants selected on kanamycin were then used to regenerate whole transgenic rape plants which though exhibiting the hairy root phenotype to varying degrees, were fertile. These plants were outcrossed with “wild type” rape lines and the F 1 progeny analysed to show segregation of hairy root and antibiotic resistance genes. In the second method, an A. tumefaciens binary system was used to transform inflorescence stalks of different varieties of rape, using modifications of existing protocols which resulted in more efficient transformation. These modifications included the use of acetosyringone, the use of N. plumbaginifolia feeder cells, the inclusion of Seaplaque agarose in the media, and the postponement of kanamycin selection in the shooting medium for 2–3 weeks. Regenerated shoots were rooted and whole rape plants produced. Putative transgenic plants were subjected to phenotypic and Southern analyses to confirm the presence or absence of the introduced genes. The methods are effective with both Spring and Winter varieties of rape. Details of the two methods are presented and the advantages and disadvantages of each method are discussed.