cry1A Gene Research Articles

Endophytic establishment of native Bacillus thuringiensis strain in maize plants and its efficacy against Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae)

BackgroundBacillus thuringiensis (Bt) is known as the most successful microbial insecticide worldwide used against lepidopteran insect pests in agriculture. Native Bacillus isolate VKK5 showing insecticidal activity against Spodoptera frugiperda (FAW) (J.E. Smith) (Lepidoptera: Noctuidae) was characterized as B. thuringiensis (BtVKK5) on a morphological and molecular basis. Recent research has shown that Bt can be established as an endophytic organism for controlling insect pests. The present work aimed at assessing the colonization of BtVKK5 as an endophyte in five maize cultivars by seed treatment (ST), soil drenching (SD), foliar application (FA) and combination of all methods (ST + SD + FA) and its bioefficacy against neonates of FAW.ResultsEstablishment of inoculated BtVKK5 as endophytes in five maize cultivars, viz. Pusa HQPM7 Improved, Pusa Jawahar Hybrid Maize 1, Pusa Vivek Hybrid 27 Improved (PVH27I), Pusa HQPM5 Improved and DMRH 1301, was confirmed by re-isolating from the leaves of the plant on ampicillin-selected agar plates. Estimation of colony-forming units per gram of leaf showed that there was a significant difference in colonization of the Bt strain among maize cultivars by different inoculation methods. The colonies were further substantiated by the amplification of cry1A and cry1E genes. Bioefficacy studies showed the highest mortality (50%) in the ST + FA + SD inoculation method, followed by ST (40%) in PVH27I. Moreover, growth inhibition was observed in survived larvae on inoculated plants vis-a-vis control.ConclusionEstablishment of Bt strain as an endophyte in maize plants, complemented with insecticidal activity, could possibly lead to an innovative approach to the management of S. frugiperda and other borers.

Comparison of the Phenotypic Performance, Molecular Diversity, and Proteomics in Transgenic Rice.

The extent of molecular diversity and differentially expressed proteins (DEPs) in transgenic lines provide valuable information to understand the phenotypic performance of transgenic crops compared with their parents. Here, we compared the differences in the phenotypic variation of twelve agronomic and end-use quality traits, the extent of microsatellite diversity, and DEPs of a recurrent parent line with three transgenic rice restorer lines carrying either CRY1C gene on chromosome 11 or CRY2A gene on chromosome 12 or both genes. The three transgenic lines had significantly smaller stem borer infestation than the recurrent parent without showing significant differences among most agronomic traits, yield components, and end-use quality traits. Using 512 microsatellite markers, the three transgenic lines inherited 2.9-4.3% of the Minghui 63 donor genome and 96.3-97.1% of the CH891 recurrent parent genome. As compared with the recurrent parent, the number of upregulated and down-regulated proteins in the three transgenic lines varied from 169 to 239 and from 131 to 199, respectively. Most DEPs were associated with the secondary metabolites biosynthesis transport and catabolism, carbohydrate transport and metabolism, post-translational modification, and signal transduction mechanisms. Although several differentially expressed proteins were observed between transgenic rice and its recurrent parent, the differences may not have been associated with grain yield and most other phenotypic traits in transgenic rice.

Abundance, distribution, and expression of nematicidal crystal protein genes in Bacillus thuringiensis strains from diverse habitats

Bacillus thuringiensis (Bt) is a Gram-positive bacterium that accumulates pesticidal proteins (Cry and Cyt) in parasporal crystals. Proteins from the Cry5, App6 (formerly Cry6), Cry12, Cry13, Cry14, Cry21, and Xpp55 (formerly Cry55) families have been identified as toxic to nematodes. In this study, a total of 846 Bt strains belonging to four collections were analyzed to determine the diversity and distribution of the Bt Cry nematicidal protein genes. We analyzed their presence by PCR, and positives were confirmed by sequencing. As a result, 164 Bt isolates (20%) contained at least one gene coding for nematicidal Cry proteins. The cry5 and cry21 genes were enriched in collection 1 and were often found together in the same strain. Differently, in collection 4, obtained from similar habitats but after 10 years, cry14 was the gene most frequently found. In collection 2, cry5 and app6 were the most abundant genes, and collection 3 had a low incidence of any of these genes. The results point to high variability in the frequencies of the studied genes depending on the timing, geographical origins, and sources. The occurrence of cry1A, cry2, and cry3 genes was also analyzed and showed that the nematicidal Cry protein genes were frequently accompanied by cry1A + cry2. The expression of the genes was assessed by mass spectrometry showing that only 14% of the positive strains produced nematicidal proteins. To our knowledge, this is the first comprehensive screening that examines the presence and expression of genes from the seven known Bt Cry nematicidal families.

Selection, effective dominance, and completeness of Cry1A.105/Cry2Ab2 dual-protein resistance in Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae)

In the U.S., Helicoverpa zea (Boddie) is a major pest targeted by both transgenic maize and cotton expressing Bacillus thuringiensis (Bt) proteins. Resistance of insect to Bt maize and cotton containing cry1A and cry2A genes has widely occurred in the U.S. In this study, two trials were performed to investigate larval survival and development of a Cry1A.105/Cry2Ab2 dual-protein resistant (VT2P-RR), a susceptible, and an F1 heterozygous (VT2P-RS) populations of H. zea on ears of nine Bt and three non-Bt maize hybrids. The Bt maize hybrids evaluated represent five common pyramided traits expressing two or three of the Cry1A.105, Cry1Ab, Cry1F, Cry2Ab2, and Vip3Aa20 proteins. In the laboratory, neonates of the three H. zea populations were inoculated on silks of ears collected from maize at R1–R2 plant stages; and larval survivorship was checked 10 d after neonate release. All three insect populations survived normally on non-Bt maize ears. Varied numbers of VT2P-RR and VT2P-RS survived on ears of Cry1A.105/Cry2Ab2 maize, while all larvae of the three populations died or could not develop on ears of Vip3Aa20-expressing maize. The results demonstrated that the dual-protein resistant H. zea was not cross-resistant to Vip3Aa20-expressing maize, and thus traits with vip3Aa20 gene should be effective to manage Cry1A.105/Cry2Ab2-resistant H. zea. The resistance in VT2P-RR was determined to be incomplete on Cry1A.105/Cry2Ab2 maize. The effective dominance levels varied greatly, from recessive to incompletely dominant, depending on maize hybrids and trials, suggesting that proper selection of maize hybrids could be important for mitigating the Cry1A.105/Cry2Ab2 resistance. The data generated should aid in modeling multiple-protein Bt resistance in H. zea.

Characterization of native Bacillus thuringiensis strains against Spodoptera litura and Spodoptera exigua

In the present study 12 native Bt strains isolated from insect cadavers were screened for their bioefficacy against neonates of economically important polyphagous pests Spodoptera litura and Spodoptera exigua by feeding assays at single concentrations of 10 μg/g of diet. Toxicity of Bt strains against neonates of S. litura and S. exigua varies from 39.04% (VKK-EV and VKK-PX2) to 70.97% (VKK-AC1) and 20.0% (VKK-AC1 and VKK-MPS) to 88.00% (VKK-AC2) on 7th day after treatment respectively. The LC50 values for potential Bt strains against S. litura varied from 0.87 μg/gm (VKK-AC1) to 9.36 μg/gm (VKK-AG2) while, against S. exigua ranged from 1.00 μg/g (VKKAC2) to 13.95 μg/g (VKK-SO) of diet. Gene profiling of potential Bt strains revealed the presence of cry1A, cry1D, cry1I, and cry2 gene. Further studies on characterization of these novel cry genes from potential native Bt strains will be valuable for management of Spodoptera spp.

Molecular cloning of a new cry2A-type gene from Bacillus thuringiensis strain Nn10 and its expression studies

In the present study, eight indigenous Bacillus thuringiensis isolates of Western Ghats of India with more than 90% toxicity against Helicoverpa armigera were characterized for cry2A gene sub families. Seven of the eight isolates harboured cry2Aa, cry2Ab and cry2Ac genes alone and or in combination. Further, the indigenous cry2Aa gene(s) from Bacillus thuringiensis isolate Nn10 which showed 100% mortality against Helicoverpa armigera was cloned and expressed into recombinant Bt strains for management of resistance development in insects. The ORF of cry2Aa (∼1.9 kb) gene(s) from Nn10 isolate was ligated with T/A vector (pTZ57 R/T) and expressed in E. coli, DH5α. Automated sequence analysis of newly cloned recombinant cry2Aa revealed 99% homology to 916 bases in the 3′ region of minus strand and 100% homology with 720 bases in the 5’ region of holotype cry2Aa1 gene. The partial Cry2Aa amino acid sequence of Bt strain, Nn10, deduced from the nucleotide sequence generated by M13F primer showed four amino acid variation in comparison to Cry2Aa1 holotype, at 338, 345, 346 and 489th position of ORF and the sequence was submitted to the NCBI. Further the expression of ORF of cry2Aa of Nn10 into acrystalliferous Bt strain, 4Q7 using expression vector pHT3P2T under the transcriptional control of cry3Aa promoter and cry2Aa terminator. SDS PAGE analysis of recombinant protein exhibited a prominent band of about 65 kDa. Bioassay studies revealed that recombinant proteins, Cry2Aa of Nn10 was toxic to Helicoverpa armigera with LC50 value of 7.26 μg ml−1.

Expression profiling of transgenes (Cry1Ac and Cry2A) in cotton genotypes under different genetic backgrounds

Transgenic cotton carrying the Cry1Ac gene has revolutionized insect pest control since its adoption, although the development of resistance in insect pests has reduced its efficacy. After 10 years of cultivating Bacillus thuringiensis (Bt) cotton with a single Cry1Ac gene, growers are on the verge of adopting Bt cotton that carries the double gene (Cry1Ac+Cry2A) due to its better effectiveness against insect pests. Thus, the current study was designed to evaluate the role of each gene in the effectiveness of Bt cotton carrying the double gene. The expression levels of the Cry1Ac and Cry2A genes were evaluated in the leaves of 10 genotypes (2 parents and 8 F1 hybrids) at 30 days after sowing (DAS), while samples of leaves, bolls and flowers were taken from the upper and lower canopies at 70 and 110 DAS. The F1 hybrids were developed through reciprocal crosses between two Bt (CKC-1, CKC-2) and two non-Bt (MNH-786, FH-942) parents. The differential expression of transgenes was evaluated through Enzyme Linked Immuno-Sorbent Assay (ELISA). The results showed that the MNH786×CKC-1 hybrid had the highest concentrations of Cry1Ac gene at 30 DAS (3.08 μg g−1) and 110 DAS (1.01 μg g−1) in leaves. In contrast, the CKC-2×MNH-786 hybrid showed the lowest concentrations of Cry1Ac gene at 30 DAS (2.30 μg g−1) and 110 DAS (0.86 μg g−1). The F1 hybrid FH-942×CKC-2 showed the highest concentrations of Cry2A gene at 30 DAS (8.39 μg g−1) and 110 DAS (7.74 μg g−1) in leaves, while the CKC-1×MNH-786 hybrid expressed the lowest concentrations of Cry2A gene at 30 DAS (7.10 μg g−1) and 110 DAS (8.31 μg g−1). A comparison between the two stages of plant growth showed that leaves had the highest concentrations at 30 DAS, whereas the lowest concentrations were observed at 110 DAS for both genes in leaves. When the expression pattern was compared between various plant parts in genotype CKC-2, it was found that leaves had higher concentrations of Cry1Ac (3.12 μg g−1) and Cry2A (8.31 μg g−1) at 70 DAS, followed by bolls (Cry1Ac (1.66 μg g−1) and Cry2A (8.15 μg g−1)) and flowers (Cry1Ac (1.07 μg g−1) and Cry2A (7.99 μg g−1)). The genotype CKC-2 had higher concentrations of Cry1Ac (3.12 μg g−1) and Cry2A (8.31 μg g−1) in the upper canopy but less accumulation (2.66 μg g−1 of Cry1Ac, 8.09 μg g−1 of Cry2A) in the lower canopy at 70 DAS. Similarly, at 110 DAS, the expression levels of Cry1Ac and Cry2A in upper and lower canopy leaves were 1.52 and 7.92 μg g−1, and 0.99 and 7.54 μg g−1, respectively. Hence, the current study demonstrates that different genotypes showed variable expression for both of the Cry1Ac and Cry2A genes during plant growth due to different genetic backgrounds. The Cry2A gene had three-fold higher expression than Cry1Ac with significant differences in expression in different plant parts. The findings of this study will be helpful for breeding insect-resistant double-gene genotypes with better gene expression levels of Cry1Ac and Cry2A for sustainable cotton production worldwide.

Enhancing the resilience of transgenic cotton for insect resistance.

The efficacy of Bt crystal proteins has been compromised due to their extensive utilization in the field. The second-generation Bt vegetative insecticidal proteins could be the best-suited alternative to combat resistance build-up due to their broad range affinity with midgut receptors of insects. The codon-optimized synthetic vegetative insecticidal proteins (Vip3Aa) gene under the control of CaMV35S promoter was transformed into a locally developed transgenic cotton variety (CKC-01) expressing cry1Ac and cry2A genes. Transformation efficiency of 1.63% was recorded. The highest Vip3Aa expression (51.98-fold) was found in MS3 transgenic cotton plant. Maximum Vip3Aa protein concentration (4.23µg/mL) was calculated in transgenic cotton plant MS3 through ELISA. The transgenic cotton plant (MS3) showed one copy number on both chromatids in the homozygous form at chromosome 8 at the telophase stage. Almost 99% mortality of H. armigera was recorded in transgenic cotton plants expressing double crystal proteins pyramided with Vip3Aa gene as contrasted to transgenic cotton plant expressing only double crystal protein with 70% mortality. The results obtained during this study suggest that the combination of Bt cry1Ac, cry2A, and Vip3Aa toxins is the best possible alternative approach to combat chewing insects.

Constitutive Expression of cry3A Gene in Transgenic Potato Plants for Resistance to Colorado Potato Beetle (CPB)

Bt crops are the most important of the commercialised transgenic plants which were developed to combat destructive pests. Colorado potato beetle (CPB) is considered as a dangerous threat to potato production. In this study transgenic potato plants expressing cry3A gene were generated using Agrobacterium mediated transformation to combat the pest damage. Polymerase chain reaction (PCR) analysis confirmed the presence of transgenes in 40 out of 110 putative transgenic lines. One to three copies of cry3A gene were identified in selected transgenic lines using southern blot analysis. Moreover, immunostrip test confirmed the expression of Cry3A protein in the leaves of transgenic potato plants. Bioassay analysis with the first instar of CPB larvae indicated 100% mortality, significantly lower leaf damage and less insect biomass accumulation for transgenic lines (PC3-3, PC3-4, PC3-5, PC3-8 and PC3-13) in comparison to control plants. The results indicated that Cry3A protein expression could efficaciously increase the resistance to CPB in transgenic potato cv. Agria which is recommended for controlling CPB damage and to prevent its spread in potato cultivation regions. Moreover, these transgenic lines can be used as cultivar or germplasm to breed new cultivars.

Growth and arthropod community characteristics of transgenic poplar 741 in an experimental forest

Use of the Bt insect-resistance gene has provided substantial economic and ecological benefits. Full utilization of transgenic poplars requires a comprehensive understanding of the growth-related phenotypic characteristics and ecological risks associated with of genetically modified trees. In the present study, we assessed the effects of transgenic poplar 741 with different insect-resistant genes on plant growth and arthropod communities in the field. Transgenic lines carrying the Cry1Ac and Cry3A genes were used as model lines, and non-transgenic poplar 741 was used as a control. Over a period of 6 years, we analyzed plant growth and transcriptomic data, and monitored arthropod richness and abundance in an experimental forest comparing transgenic and control lines. Our results indicate that the Pb29 line had a similar plant height to CK, but a significantly lower diameter at breast height (DBH) than CK. The plant height and DBH of the CC84 line were slightly higher than those of CK, but the differences were not significant. Transcriptome data analysis showed downregulated and upregulated expression of growth hormone-related genes and lignin synthesis-related genes, respectively, in the Pb29 line compared with CK and the CC84 line. The survey data obtained in this study showed that annual trends in arthropod numbers in the ecosystem were consistent among transgenic poplar 741 and control lines, gradually decreasing with time since planting. The Cry1Ac line has selective resistance to Lepidoptera and exerted strong toxic effects on Arctiidae, Tortricidae, and Notodontidae, but had no obvious effects on Lyonetiidae or Geometridae. The Cry3A line exhibited significant resistance to coleopterans only in 2016 when the abundance of coleopteran insects was high. Both lines exhibited inhibitory effects on non-target pests. The two transgenic lines were similar to the control with respect to arthropod community structure, and there were no significant differences in arthropod diversity indices between transgenic and control lines, although richness was slightly higher for the Cry3A line than for the control. The results of this study showed that the insertion of Bt genes had no apparent inhibitory effect on the growth of poplar 741. Also, there was no adverse environmental impact of transgenic poplar 741 on ecosystem arthropod communities compared with conventional poplar 741.

Transformation and evaluation of Broad-Spectrum insect and weedicide resistant genes in Gossypium arboreum (Desi Cotton)

ABSTRACT Gossypium arboreum (Desi Cotton) holds a special place in cotton industry because of its inherent ability to withstand drought, salinity, and remarkable resistance to sucking pests and cotton leaf curl virus. However, it suffers yield losses due to weeds and bollworm infestation. Genetic modification of G. arboreum variety FBD-1 was attempted in the current study to combat insect and weedicide resistance by incorporating cry1Ac, cry2A and cp4-EPSPS genes under control of 35S promoter in two different cassettes using kanamycin and GUS as markers through Agrobacterium-mediated shoot apex cut method of cotton transformation. The efficiency of transformation was found to be 1.57%. Amplification of 1700 bp for cry1Ac, 167 bp for cry2A and 111 bp for cp4-EPSPS confirmed the presence of transgenes in cotton plants. The maximum mRNA expression of cry1Ac and cp4-EPSPS was observed in transgenic cotton line L3 while minimum in transgenic cotton line L1. The maximum protein concentrations of Cry1Ac, Cry2A and Cp4-EPSPS of 3.534 µg g−1, 2.534 µg g−1 and 3.58 µg-g−1 respectively were observed for transgenic cotton line L3 as compared to control cotton line. On leaf-feed-based insect bioassay, almost 99% mortality was observed for Helicoverpa armigera on the transgenic cotton plant (L3). It completely survived the 1900 ml hectare−1 glyphosate spray assay as compared to non-transgenic cotton plants. The necrotic spots appeared on the third day, leading to the complete death of control plants on the fifth day of assay. The successful multiple gene-stacking in G. arboreum FBD-1 variety could be further used for qualitative improvement of cotton fiber through plant breeding techniques.

Food/Feed and Environmental Risk Assessment of Insect-resistant Genetically Modified Maize MIR604 for Food and Feed Uses, Import and Processing under Regulation (EC) No 1829/2003 (EFSA/GMO/UK/2005/11)

Food/Feed and Environmental Risk Assessment of Insect-resistant Genetically Modified Maize MIR604 for Food and Feed Uses, Import and Processing under Regulation (EC) No 1829/2003 (EFSA/GMO/UK/2005/11)

Comparing the Immunity of Genetically and Non-Genetically Modified Rice regarding the Possibility of Gene Transferring to Blood of a Laboratory Animal

Introduction: Genetically modified plant species are produced by the genetic engineering of agricultural science and the addition of specific genes in their genetic sequences with the aim of optimizing the product and creating desirable traits. This study examined the remains of Cry1A(b) and P35 genes in blood of rats fed with genetically modified rice. Methods: This experimental and interventional study was conducted on male and female rats Sprague Dawley (SD), which were divided into treatment and control groups according to the type of food received. Each group consisted of 10 rats (5 male and 5 female); the first group consumed 50 percent of the genetically modified rice and the second group consumed 50percent of non- genetically modified rice from the total needed carbohydrate. Blood sampling and DNA extraction were conducted after 90 days of feeding rats with nutritional pattern using a kit. Quantitative and qualitative tests were carried out on the extracted DNA using agarose gel electrophoresis and spectrophotometry, respectively. Results: Agarose gel electrophoresis was used and the results showed no transgenic genes in the studied blood samples. Conclusion: The results showed no significant difference between the the treatment and control rats with regard to the presence of transgenic genes of p35 and Cry1A(b). Therefore, the possibility of gene transfer to the organs of rats consuming genetically modified rice was rejected. Results of this study showed no difference between the safety of genetically-modified and non- modified rice with regard to gen transferring.

Molecular characterization of lepidopteran-specific toxin genes in Bacillus thuringiensis strains from Thailand.

A total of 511 local isolates of Bacillus thuringiensis from different geographical regions of Thailand were analyzed for the presence of the cry1A, cry1B, cry2A, cry9, and vip3A genes encoding for lepidopteran-specific toxins. PCR results revealed that 94.32% (482/511) of B. thuringiensis isolates harbored at least one of the detected genes, of which the cry1A, cry1B, cry2A, cry9, and vip3A genes were detected at frequencies of 90.61%, 89.63%, 76.32%, 40.70%, and 48.18%, respectively. Nineteen gene-combination profiles were discovered among 482 B. thuringiensis isolates, of which the most frequently detected profile contained the cry1A, cry1B, cry2A, and vip3A genes. Sixty-one isolates (12.66%), which harbored all of the detected insecticidal toxin genes, were further detected for the exochitinase (chi36) gene and chitinase activity. The results revealed that all 61 isolates contained the chi36 gene and exhibited chitinase activity. Insect bioassays showed that five isolates were highly toxic (more than 80% mortality) against second instar larvae of Spodoptera litura, of which the highest insect mortality (93%) was obtained from the B. thuringiensis isolates 225-15 and 417-1. Scanning electron microscopy revealed that the crystal morphologies of the five effective isolates were bipyramidal and cuboidal shapes. SDS-PAGE analysis of the spore-crystal mixture showed major bands of approximately 65 and 130kDa. These five effective strains are alternative candidates for use as a microbial insecticide for the control of the S. litura pest.

Determination of cry2A genes in Bacillus thuringiensis isolated from southern provinces of Vietnam

In 27 isolates of Bacillus thuringiensis from southern provinces of Vietnam with ability to produce bipyramidal inclusions, 21 isolates were positive for cry2A-type genes via PCR methods. Analysis of DNA sequences revealed the amplified products belonged to cry2A-type genes of B. thuringiensis with identity above 90% compared with cry2A sequences published on GenBank sequence database. The identity values of the amplified products of the isolates BT5 and TN7.1 with cry2A group primers were 97% and 99%, respectively, while the identity of 99% were observed in the PCR products of TN7.1 with specific primers for partial sequences of cry2Aa and cry2Ab. These results may be useful for prediction of insecticidal activity of the B. thuringiensis isolates and development of new bioinsecticide products.

Molecular characterization of an indigenous lepidopteran toxic Bacillus thuringiensis strain T532

Bacillus thuringiensis Berliner (Bt) being an eco-friendly bioinsecticide is effectively used in pest management strategies and, therefore, isolation and identification of new strains effective against a broad range of target pests is important. In the present study, four indigenous Bt isolates (T489, T491, T495 and T532) were taken and investigated for their properties such as crystal morphology, cry gene(s) content and insecticidal activity. Bipyramidal, cuboidal and spherical crystals were observed. Among the four isolates only one isolate was found positive for lepidopteran specific cry genes. The isolate T532 which showed the presence of cry1, cry2Aa and cry2Ab genes was selected for protein profiling and bioassay. The isolate showed the presence of two proteins of molecular weights at ~135 and ~65 kDa sizes. Artificial diet based bioassay resulted in 100% mortality of the neonate larvae of H. armigera and S. litura for the isolate T532 which was comparable to that of the positive control (HD1). The full length cry2A gene was amplified and cloned into E. coli DH5α strain. The recombinant plasmids were sequenced and the sequence was deposited in GenBank nucleotide database. The accession number of the gene is MH475905. The gene was named by the Bacillus thuringiensis delta endotoxin nomenclature committee as cry2Aa23 .

Particle Bombardment of the cry2A Gene Cassette Induces Stem Borer Resistance in Sugarcane.

Sugarcane borer is the most common and harmful pest in Chinese sugarcane fields, and can cause damage to the whole plant during the entire growing season. To improve borer resistance in sugarcane, we constructed a plant expression vector pGcry2A0229 with the bar gene as the marker and the cry2A gene as the target, and introduced it into embryogenic calli of most widely cultivated sugarcane cultivar ROC22 by particle bombardment. After screening with phosphinothricin in vitro and Basta spray, 21 resistance-regenerated plants were obtained, and 10 positive transgenic lines harboring the cry2A gene were further confirmed by conventional PCR detection. Real-time quantitative PCR (RT-qPCR) analysis showed that the copy number of the cry2A gene varied among different transgenic lines but did not exceed four copies. Quantitative ELISA analysis showed that there was no linear relationship with copy number but negatively correlated with the percentage of borer-infested plants. The analysis of industrial and agronomic traits showed that the theoretical sugar yields of transgenic lines TR-4 and TR-10 were slightly lower than that of the control in both plant cane and ratoon cane; nevertheless, TR-4 and TR-10 lines exhibited markedly lower in frequency of borer-infested plants in plant cane and in the ratoon cane compared to the control. Our results indicate that the introduction of the cry2A gene via bombardment produces transgenic lines with obviously increased stem borer resistance and comparable sugar yield, providing a practical value in direct commercial cultivation and crossbreeding for ROC22 has been used as the most popular elite genitor in various breeding programs in China.

Development of transgenic tobacco lines with pyramided insect resistant genes.

Insect pests are among the major constraints rendering drastic decreases in crop yield. The expression of stacked insecticidal genes in crops can lead to resistance durability and can delay the development of resistance in target insect pests. The present study was designed to introduce an insect resistance trait in locally cultivated Turkish tobacco cultivars (Basma and Nail) with pyramided insecticidal genes. Agrobacterium strain LBA4404 harboring plasmid pKGH4 with cry1Ac and cry2A genes under the control of 35S promoter was used to infect leaf discs of both cultivars; plasmid also contained uidA within the T-DNA region for earlier screening of putative transformants. The overall transformation efficiency was calculated as 30.7% and 18.8% in Basma and Nail, respectively. PCR results confirmed the integration of cry1Ac, cry2A, uidA, and nptII genes in 40 plants of Basma and 16 plants of Nail. ELISA results showed variation in expression of cry1Ac protein among transgenic plants varying from 0.017 to 0.607 µg/g of fresh tissue. Bioassay results with potato tuber moth (Phthorimea operculella Zeller) showed significant mortality of the targeted pest on primary transformants. Furthermore, T1 transgenic progeny exhibited the inheritance of T-DNA in Mendelian as well as non-Mendelian fashion. The results revealed that lines can serve as a source of germplasm in tobacco breeding programs.

Tomato CRY1a plays a critical role in the regulation of phytohormone homeostasis, plant development, and carotenoid metabolism in fruits.

Blue light photoreceptors, cryptochromes (CRYs), regulate multiple aspects of plant growth and development. However, our knowledge of CRYs is predominantly based on model plant Arabidopsis at early growth stage. In this study, we elucidated functions of CRY1a gene in mature tomato (Solanum lycopersicum) plants by using cry1a mutants and CRY1a-overexpressing lines (OE-CRY1a-1 and OE-CRY1a-2). In comparison with wild-type plants, cry1a mutants are relatively tall, accumulate low biomass, and bear more fruits, whereas OE-CRY1a plants are short stature, and they not only flower lately but also bear less fruits. RNA-seq, qRT-PCR, and LC-MS/MS analysis revealed that biosynthesis of gibberellin, cytokinin, and jasmonic acid was down-regulated by CRY1a. Furthermore, DNA replication was drastically inhibited in leaves of OE-CRY1a lines, but promoted in cry1a mutants with concomitant changes in the expression of cell cycle genes. However, CRY1a positively regulated levels of soluble sugars, phytofluene, phytoene, lycopene, and ß-carotene in the fruits. The results indicate the important role of CRY1a in plant growth and have implications for molecular interventions of CRY1a aimed at improving agronomic traits.

Development and relative fitness of Cry1C resistance in Chilo suppressalis.

Chilo suppressalis is an important lepidopteran rice pest in the rice-growing areas of China, and the development of transgenic rice expressing the Cry1C insecticidal protein has provided a useful strategy for controlling this pest. However, insect resistance is a major threat to the durability of cry1C rice. Thus, evaluation of the risk of insect resistance before the commercial use of cry1C rice is crucial. This study investigated the development of C. suppressalis resistance to Cry1C protein and the relative fitness of Cry1C-resistant and -susceptible strains on different Bt rice lines. The LC50 value of the Cry1C-resistant strain increased 42.6-fold after 41 generations of selection, and the estimated realized heritability (h2 ) of Cry1C resistance was 0.096 in C. suppressalis. Moreover, the Cry1C-resistant strain displayed high fitness on the cry1C line, but not on the cry1Ab and cry1Ab + cry1C lines and was not cross-resistant to Cry1Ab. These findings suggest that C. suppressalis has the potential to develop resistance to Cry1C, although the rate of evolution is low. The pyramiding of the cry1A and cry1C genes in Bt rice is an effective strategy for delaying the evolution of resistance in C. suppressalis and sustainably maintaining the utility of Bt rice. © 2017 Society of Chemical Industry.