Stable expression of exogenous imported <Emphasis Type="Italic">sporamin</Emphasis> in transgenic Chinese cabbage enhances resistance against insects

Cultivating insect pest-resistant varieties is one of the most effective ways to prevent or mitigate pest infestation in Chinese cabbage (Brassica campestris ssp. chinensis). Via the agrobacterium tumefaciens-mediated transformation method, this study introduced the protease inhibitor encoding gene sporamin into two widely cultured cultivars ‘Youdonger’ and ‘Shanghaiqing’, of the common variety of Chinese cabbages (B. campestriss ssp. chinensis var. communis), getting transgenic plants with high sporamin expression. In vitro insect bioassays indicated that, compared with the wild type plants, the transgenic plants exhibited improved resistance to diamondback moth (Plutella xylostella L.) The analysis of inheritance pattern of exogenous sporamin in the progenies of single copy insertion transgenic lines demonstrated that sporamin could be inherited and expressed stably in transgenic progenies. Field survey of the insect resistance under the normal culture condition confirmed the enhanced resistance in transgenic progenies to diamondback moth. Our results strongly suggest that sporamin is an efficient candidate gene for insect-resistant genetic engineering in Chinese cabbage.     

Greenhouse and field evaluations of transgenic canola against diamondback moth, shape Plutella xylostella, and corn earworm, shape Helicoverpa zea

Canola (Brassica napus L.) cultivars Oscar and Westar, engineered with a Bacillus thuringiensis (Bt) cryIA(c) gene, were evaluated for resistance to lepidopterous pests, diamondback moth, Plutella xylostella L. (Plutellidae) and corn earworm, Helicoverpa zea (Boddie) (Noctuidae) in greenhouse and field conditions. In greenhouse preference assays conducted at vegetative and flowering plant stages, transgenic plants recorded very low levels of damage. A 100% diamondback moth mortality and 90% corn earworm mortality were obtained on transgenic plants in greenhouse antibiosis assays. The surviving corn earworm larvae on transgenic plants had reduced head capsule width and body weight. Mortality of diamondback moth and corn earworm were 100% and 95%, respectively, at different growth stages (seedling, vegetative, bolting, and flowering) on the transgenic plants in greenhouse tests. In field tests conducted during 1995–1997, plots were artificially infested with neonates of diamondback moth or corn earworm or left for natural infestation. Transgenic plants in all the treatments were highly resistant to diamondback moth and corn earworm larvae and had very low levels of defoliation. Plots infested with diamondback moth larvae had greater damage in both seasons as compared with corn earworm infested plots and plots under natural infestation. After exposure to defoliators, transgenic plants usually had higher final plant stand and produced more pods and seeds than non-transgenic plants. Diamondback moth injury caused the most pronounced difference in plant stand and pod and seed number between transgenic and non-transgenic plants. Our results suggest that transgenic canola could be used for effective management of diamondback moth and corn earworm on canola.     

Characterization of a myrosinase cDNA from Brassica parachinensis and its defense role against Plutella xylostella after suppression

Abstract In Brassicaceae, myrosinase catalyzes the hydrolysis of glucosinolate and plays an important role in anti‐herbivore defense. We have cloned and characterized the full‐length complementary DNA of myrosinase gene from Brassica parachinensis that exhibits high sequence identity with myrosinase genes from other Brassica species. To investigate the role of this myrosinase in defense against the diamondback moth (Plutella xylostella), we constructed an RNA‐interference (RNAi) cassette expressing a double‐stranded RNA that targeted myrosinase and transfected it into B. parachinensis. Myrosinase was suppressed in the resulting transgenic plants. Diamondback moth larvae feeding on transgenic plants had lower larval and pupal weights, longer pupal duration, and lower fecundity than those feeding on non‐transgenic plants, suggesting that the diamondback moth has adapted to the glucosinolate‐myrosinase defensive system. Therefore, the suppression of myrosinase is a potential approach for controlling the diamondback moth.     

Insect-resistant transgenic Pinus radiata

Transgenic radiata pine (Pinus radiata D. Don) plants containing a Bacillus thuringiensis (Bt) toxin gene, crylAc, were produced by means of biolistic transformation of embryogenic tissue. Using the selectable marker gene nptII and corresponding geneticin selection, 20 independent transgenic lines from five genotypes were established. Over 200 plants regenerated from ten transgenic lines were successfully transferred to soil. The integration and expression of the introduced genes in transgenic tissue and/or plants were confirmed by PCR, Southern hybridisation and neomycin phosphotransferase II (NPTII) and Bt ELISA assays. Bioassays with larvae of the painted apple moth, Teia anartoides, demonstrated that transgenic plants displayed variable levels of resistance to insect damage, with one transgenic line being highly resistant to feeding damage.     

Resistance to Tecia solanivora (Lepidoptera: Gelechiidae) in three transgenic Andean varieties of potato expressing Bacillus thuringiensis CrylAc protein

Transgenic potato, Solanum tuberosum L., plants containing a synthetic cry1Ac gene coding for the Bacillus thuringiensis (Bt) crystalline insecticidal protein were produced and evaluated for resistance to Tecia solanivora Povolny (Lepidoptera: Gelechiidae), the larvae of which attack potato tubers. In total, 43 transgenic lines of commercial Andean potato varieties Diacol Capiro, Pardo Pastusa, and Pandeazúcar were obtained. These transgenic lines were found to have one to four copies of cry1Ac per genome and expression levels of Cry1Ac protein varying from 0.02 to 17 microg/g fresh tuber tissue. Bioassays of T. solanivora larvae on these transgenic potato tubers showed 83.7-100% mortality, whereas the mortality levels on nontransgenic lines were 0-2.67%. Our data indicate the capability of Bt transgenic technology to control the T. solanivora while reducing the use of chemical insecticides. Further studies under controlled field conditions will be helpful in exploring the potential of CrylAc potatoes in the insect pest management strategies.     

Wound-response regulation of the sweet potato sporamin gene promoter region

Sporamin, a tuberous storage protein of sweet potato, was systemically expressed in leaves and stems by wound stimulation. In an effort to demonstrate the regulatory mechanism of wound response on the sporamin gene, a 1.25 kb sporamin promoter was isolated for studying the wound-induced signal transduction. Two wound response-like elements, a G box-like element and a GCC core-like sequence were found in this promoter. A construct containing the sporamin promoter fused to a -glucuronidase (GUS) gene was transferred into tobacco plants by Agrobacterium-mediated transformation. The wound-induced high level of GUS activity was observed in stems and leaves of transgenic tobacco, but not in roots. This expression pattern was similar to that of the sporamin gene in sweet potatoes. Exogenous application of methyl jasmonate (MeJA) activated the sporamin promoter in leaves and stems of sweet potato and transgenic tobacco plants. A competitive inhibitor of ethylene (2,5-norbornadiene; NBD) down-regulated the effect of MeJA on sporamin gene expression. In contrast, salicylic acid (SA), an inhibitor of the octadecanoid pathway, strongly suppressed the sporamin promoter function that was stimulated by wound and MeJA treatments. In conclusion, wound-response expression of the sporamin gene in aerial parts of plants is regulated by the octadecanoid signal pathway.     

Transgenic broccoli expressing aBacillus thuringiensis insecticidal crystal protein: Implications for pest resistance management strategies

We usedAgrobacterium tumefaciens to transform flowering stalk explants of five genotypes of broccoli with a construct containing the neomycin phosphotransferase gene and aBacillus thuringiensis (Bt) gene [CryIA(c) type] optimized for plant expression. Overall transformation efficiency was 6.4%; 181 kanamycin-resistant plants were recovered. Of the 162 kanamycin-resistant plants tested, 112 (69%) caused 100% morality of 1st-instar larvae of aBt-susceptible diamondback moth strain. Southern blots of some resistant transformants confirmed presence of theBt gene. Selected plants that gave 100% mortality of susceptible larvae allowed survival of a strain of diamondback moth that had evolved resistance toBt in the field. F₁ hybrids between resistant and susceptible insects did not survive. Analysis of progeny from 26 resistant transgenic lines showed 16 that gave segregation ratios consistent with a single T-DNA integration. Southern analysis was used to verify those plants possessing a single T-DNA integration. Because these transgenic plants kill susceptible larvae and F₁ larvae, but serve as a suitable host for resistant ones, they provide an excellent model for tests ofBt resistance management strategies.     

Improved tolerance against <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> in transgenic tomato over-expressing multi-domain proteinase inhibitor gene from <Emphasis Type="Italic">Capsicum annuum</Emphasis>

Plant proteinase inhibitors (PIs) are plant defense proteins and considered as potential candidates for engineering plant resistances against herbivores. Capsicum annuum proteinase inhibitor (CanPI7) is a multi-domain potato type II inhibitor (Pin-II) containing four inhibitory repeat domains (IRD), which target major classes of digestive enzymes in the gut of Helicoverpa armigera larvae. Stable integration and expression of the transgene in T1 transgenic generation, were confirmed by established molecular techniques. Protein extract of transgenic tomato lines showed increased inhibitory activity against H. armigera gut proteinases, supporting those domains of CanPI7 protein to be effective and active. When T1 generation plants were analyzed, they exhibited antibiosis effect against first instar larvae of H. armigera. Further, larvae fed on transgenic tomato leaves showed delayed growth relative to larvae fed on control plants, but did not change mortality rates significantly. Thus, better crop protection can be achieved in transgenic tomato by overexpression of multi-domain proteinase inhibitor CanPI7 gene against H. armigera larvae.     

Transgenic <Emphasis Type="Italic">Brassica napus</Emphasis> L. lines carrying a two gene construct demonstrate enhanced resistance against <Emphasis Type="Italic">Plutella xylostella</Emphasis> and <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

Plant diseases and insect pests are serious threat to the growth and yield of oilseed rape. In this study, a binary vector carrying sporamin and chitinase PjChi-1 genes in tandem was introduced into Brassica napus cv. ZS 758 via Agrobacterium tumefaciens for dual resistance against disease and insect attack. Thirty-two regenerated plantlets exhibiting hygromycin resistance were selected following Agrobacterium-mediated transformation of 600 leaf petiole explants. Of these, 27 transformants were confirmed to carry the two transgenes as detected by polymerase chain reaction (PCR) with 4.5% transformation efficiency. Eight plantlets were randomly selected for further confirmation by Southern and northern blot hybridization analyses. Four plants carried single copy of the transgenes, while the remaining four plants carried either two or three copies of the transgenes. Moreover, expression of the sporamin transgene was detected by northern blot hybridization in transgenic lines, but not in wild-type plants. These eight T₀ plants were grown in vitro, and inoculated with the Lepidoptera larvae of Plutella xylostella and with spores of the fungal pathogen of Sclerotinia sclerotiorum. Transgenic plants exhibited high levels of resistance to P. xylostella and S. sclerotiorum when compared to untransformed wild-type plants. Genetic analysis of T₁ progeny confirmed Mendelian segregation of the introduced genes. Therefore, these transgenic lines demonstrate a promising potential for variety development of oilseed rape lines with enhanced resistance against both P. xylostella and S. sclerotiorum.     

Stability of sheath blight resistance in transgenic ASD16 rice lines expressing a rice <Emphasis Type="Italic">chi11</Emphasis> gene encoding chitinase

Development of transgenic plants by introducing defense genes is one of the strategies to engineer disease resistance. Transgenic ASD16 rice plants harbouring rice chitinase chi11 gene, belonging to a PR-3 group of defense gene conferring sheath blight (Rhizoctonia solani Kuhn) resistance, were used in this study. Three T₂ homozygous lines (ASD16-4-1-1, 5-1-1, and 6-1-1) were identified from seven putative (T₀) transgenic lines expressing chi11 using Western blotting analysis. The inheritance of sheath blight resistance in those lines was studied over generations. The stability of chi11 expression up to T₄ generation in all the three homozygous lines was proved by Western blot and the stability of sheath blight resistance in the homozygous lines was proved up to T₄ generation using detached leaf and intact leaf sheath assays. Among the three homozygous lines tested, ASD16-4-1-1 showed consistent results in all the generations and gave a better protection against the sheath blight pathogen than the other two lines.     

Genetic transformation of potato with nptII-gus marker genes enhances foliage consumption by Colorado potato beetle larvae

Little is known about the effect of transgenic plants containing commonly used marker genes, such as aph(3)II (nptII encoding neomycinphosphotransferase) and uidA (gus encoding -glucuronidase) on insect feeding behaviour. We report here, for the first time, that transgenic potato plants containing only nptII and gus marker genes enhance foliage consumption by the Colorado potato beetle (CPB, Leptinotarsa decemlineata S.). Transformation of potato cultivar Désirée was performed with Agrobacterium tumefaciens. Internode explants were inoculated with different strains of bacteria, carrying either nptII-gus or nptII alone. A total of 180 transgenic and untransformed control plants were grown in the greenhouse for the analysis of food consumption by CPB. For each transformed and untransformed line tested, four bioassays were conducted each consisting of 10 second-instar larvae feeding independently on a 2 cm diameter leaf disc for 20 h. Our data show up to 50% increase of mean foliage consumption on plants transformed with the nptII-gus construct, indicating that transgenic plants containing these marker genes can affect the feeding behaviour of the insects. These results were obtained from the primary regenerants (R₀ lines) as well as from tuber-derived plants (R₁ lines). Further tests with transgenic plants containing the nptII marker gene only, showed no significant difference in feeding when compared to untransformed control plants, allowing us to rule out a direct effect of this marker gene on foliage consumption by the insect larvae. It is suggested that gus protein is involved in the increase of foliage consumption by CPB.     

Effect of Bt maize on the reproduction and development of saprophagous Diptera over multiple generations

A laboratory experiment was used to quantify the effects of Bt maize on Drosophila melanogaster and Megaselia scalaris, representatives of two saprophagous dipteran families (Drosophilidae, Phoridae). Freshly hatched larvae were reared on a diet containing decaying maize leaves. Two transgenic maize varieties, expressing Cry3Bb1 or Cry1Ab, and their corresponding isolines were tested. In an additional treatment, a solution of pure Cry1Ab was added to the maize diet. According to quantitative ELISA analyses, all Bt diets and all larvae feeding on Bt maize contained low concentrations of Cry proteins but Cry proteins were not detected in adults, thus, predators of the larvae are exposed to Cry proteins whereas predators of adult flies are not. Highest concentrations were in larvae feeding on a maize diet supplemented with a Cry1Ab protein solution. The developmental time and fertility (offspring/female) were measured over four generations for D. melanogaster and over three generations for M. scalaris. Only a few significant differences were found between transgenic and non-transgenic treatments but the differences were not consistent and did not indicate any negative effects of Bt proteins. We conclude that D. melanogaster and M. scalaris larvae are not affected in the long term when feeding and developing on decaying Cry1Ab and Cry3Bb1 maize leaves.     

Effect of pea lectin expressed transgenically in oilseed rape on pollen beetle life-history parameters

When investigating resistance to herbivorous insects in transgenic plants, it is primarily the damaging stage of the insect that is targeted. In our study, we showed that small effects of the transgenic plant on the pest performance might lead to a clear effect on the pest population level. Pollen beetle, Meligethes aeneus (Fabricius) (Coleoptera: Nitidulidae), larvae were reared on three transgenic lines of Brassica napus L. (Brassicaceae) expressing pea (Pisum sativum L.) lectin at 0.2, 0.6, and 0.7% of total soluble protein in anthers, and then monitored until the adult stage. Two lines without the gene construct and consequently containing no pea lectin were used as controls. The egg viability, larval weight, development time, and survival rate were recorded. In a separate experiment, the development time from egg to adult, adult weight, and winter survival of pollen beetles developed on intact plants were recorded. Survival of larvae to adult stage was significantly lower on the transgenic plant lines, independent of lectin level. Survival during hibernation was only 2.4% and was not affected by plant line. Combining previously published results from adult feeding and oviposition assays with the new data on larval development we summarize the effect of the transgenic oilseed rape on the pollen beetle, from oviposition in spring to overwintered adults of the new generation. Net reproductive rate was then shown to be reduced by half on the transgenic oilseed rape compared to the control plants.     

Overexpression of Pp14-3-3 from Pyrus pyrifolia fruit increases drought and salt tolerance in transgenic tobacco plant

Drought and salinity are the major abiotic stresses, which reduce agricultural productivity. In plants, 14-3-3s function as regulators of many target proteins through direct protein-protein interactions and play an important role during response to abiotic stresses. Here we report that CaMV 35S promoter driven overexpression of a Pyrus pyrifolia 14-3-3 gene, Pp14-3-3, improves drought and NaCl tolerance in T1 generation plants of transgenic tobacco (Nicotiana tabacum L. cv Xanthi). Under drought and NaCl stresses, the Pp14-3-3 was largely expressed in T1 transgenic tobacco lines, and compared with the wild-type (WT), transgenic tobacco plants showed relatively normal growth condition. In addition, the levels of membrane lipid peroxidation in T1 transgenic lines were definitely lower than that in WT according to the significantly decreased content of malondialdehyde. Meanwhile, the T1 transgenic tobacco lines showed significantly slower superoxide anion production rate than the WT under abiotic stress. Moreover, both the glutathione S-transferase (GST) and ascorbate peroxidase (APX) activities in T1 transgenic lines were markedly higher than those in WT. GSTs and APXs are important components of plant antioxidant system, and the results of present study suggested that Pp14-3-3 should play a crucial role in reducing oxidative damage caused by drought and salt stresses.     

Expression of a Bacillus thuringiensis cry1Ab gene in transgenic white spruce and its efficacy against the spruce budworm (Choristoneura fumiferana)

A synthetic version of the cry1Ab gene from Bacillus thuringiensis (Bt) was introduced into white spruce (Picea glauca) by microprojectile bombardment. A plasmid carrying the cry1Ab gene, driven by a ubiquitin (maize) promoter, was co-transferred with a plasmid containing the gus–nptII fusion gene as a screenable selection marker. Molecular analysis of the transgenic lines showed a high level (more than 90%) of co-integration of the cry1Ab gene with the screenable marker. A wide range of expression levels of the cry1Ab gene and corresponding endotoxin was obtained. Accumulation of the Cry1Ab protein was evaluated in embryogenic tissue, the needles of somatic seedlings and in the needles of 5-year-old field-grown trees of individual lines. Laboratory and field insect feeding trials suggest that several spruce transgenic lines were lethal to spruce budworm larvae.