Bt Gene Research Articles

Transgenic tobacco expressing a novel Bt gene, cry1AcF, show resistance against fall armyworm (Spodoptera frugiperda)

Transgenic tobacco expressing a novel Bt gene, cry1AcF, show resistance against fall armyworm (Spodoptera frugiperda)

Application and Development of Bt Insect Resistance Genes in Rice Breeding

As pests are an important factor in reducing crop yields, pest control is an important measure in preventing reductions in crop yields. With the aim of ending the use of chemical pesticides, biological control and genetically modified methods are now considered more reasonable pest control strategies. The bacterium Bacillus thuringiensis (Bt) can produce crystal proteins that have specific toxicity to lepidopteran insects, and so it has been applied as a microbial insecticide in the control of crop pests for several decades. With the development of plant genetic engineering, Bt genes encoding insecticidal crystal protein have been introduced into many crop species for pest control. This article indicates that, after years of experiments and research, Bt transgenic rice is close to becoming a commercial insect-resistant rice, and many studies have shown that transgenic rice has pronounced abilities in the control of pests such as yellow stem borers (Scirpophaga incertulas, YSB), striped stem borers (Chilo suppressalis, SSB), and rice leaf rollers (Cnaphalocrocis medinalis, RLR); moreover, it does not obviously differ from non-transgenic rice in terms of safety. This paper suggests that transgenic Bt rice has application potential and commercial value.

Comparison of cotton (Gossypium hirsutum) hybrids and their non-transgenic in India

It is still not clear whether differential response of Bt cotton and conventional non-Bt cotton genotypes in terms of growth, phenology and yield ability are hypothetical or realistic. This study investigated the response of three Bt cotton hybrids in comparison to their conventional non-transgenic version (near-isogenic lines) along with two locally adapted genotypes for growth, yield attributes, phenological characteristics and rooting behavior. Therefore, two experiments were conducted during summer season of year 2016 and 2017 to evaluate differences among 3 popular Bt cotton hybrids along with their non-Bt version. First field experiment was conducted as a pot study wherein shoot and root parameters of 3 Bt cotton hybrids (Ankur 3028, MRC 7017, Bioseed 6588) were compared with their non-Bt version along with 2 local checks (i.e.,hirsutum cotton Cv. F2228 and arboreum cottonCv. FDK124). Second field experiment evaluated growth, phenological characteristics, yield attributes and productivity potential of above mentioned Bt and non-Bt hybrids. Findings of pot experiment could not establish any significant difference among studied Bt and non-Bt hybrids for root and shoot characteristics. However, arboreum cotton FDK124 exhibited higher root length than all genotypes. Results of second experiment revealed significant differences for phenological characteristics, wherein Bt version of all the tested hybrids exhibited earliness for flowering initiation, 50 % flowering and days to maturity over their non-Bt version. Seed cotton yield (SCY) in all hybrids possessing Bt gene was significantly higher than their non-Bt version primarily due to higher bolls plant-1. Studies conclude that introgression of Bt gene could not alter rooting parameters of evaluated cotton genotypes but significantly affected phenological stages by inducing earliness and higher SCY in Bt versions due to improved bolls plant-1. Keywords: Bt cotton; Near-isogenic lines; Phenology; Root dry matter; Seed cotton yield

Efficacy of drought-tolerant and insect-protected transgenic TELA® maize traits in Nigeria.

Assessment of efficacy of drought tolerance (DT) and insect protection (Bt) genes in maize genotypes is invaluable for commercialization and production of transgenic maize in Nigeria. Seven maize hybrids, known as TELA® maize, with stacked events of Bt insect protection (MON89034) and drought tolerance (MON87460; DroughtGard®) and their respective non-GM versions (isohybrids) developed through the TELA Maize Project were evaluated in confined field trial site at Zaria in 2020 and 2021. The objective was to assess the efficacy of stacked DT and Bt genes to seek deregulation and commercialization of both traits in Nigeria. Significant (P < 0.05-0.01) differences were observed among genotypes (G), environments (E) and genotype × environment interaction (GEI) for grain yield and most other traits under stem borer (moth species) and fall armyworm infested, drought stress, and optimum-moisture conditions, except E and GEI under drought. TELA® GM hybrids with Bt MON89034 had 19% higher yield than their non-GM isogenic versions, and 40% higher yield than the commercial checks under the target pests infestation. The foliar damage score of all the TELA® GM genotypes was ≤ 2 relative to their non-GM isogenic versions which scored ≥ 4, indicating the effectiveness of the Bt MON89034 gene in conferring resistance against stem borer and fall armyworm. Under moderate drought, pairwise comparison showed TELA® GM Hybrid 1-1 and Hybrid 2-1 had 12.4-20.4% higher (P < 0.01) yield than their isogenic versions. Under optimum-moisture condition with pests controlled, the TELA® GM and their isogenic hybrids were similar, but both had 32% higher yield than the commercial checks. Adoption of TELA® GM technology by farmers as adaptation strategy to cope with climate change, will ensure sustainability of maize production and productivity in Nigeria.

Detection of Smuggled Genetically Modified Crops and Assessment of its Environmental Impact in the Ethio -Sudan Trans Boundary Area, North West Ethiopia

Background The genetic material of the genetically modified crop has been altered to develop the necessary insect resistance features by introducing genes from the Bt (Bacillus thuringiensis) bacterium. The objective of this study was to find smuggled GM Bt crops in the Metema farming area and examine its environmental effects. Method An experimental; Completely Randomized Design (CRD) was used to collect crop samples in the study area. The CTAB (Cetyltrimethyl ammonium bromide) technique was used to isolate DNA from all transported samples, and the purity was determined using a Nano Drop spectrophotometer. Conventional PCR with particular primers for different Bt gene events was used to detect the presence of genes. Furthermore, utilizing Bt cotton specific primer sets, the prevalence of GM cotton was measured, and amplified fragments were confirmed using agarose gel electrophoresis. Result The PCR results revealed that 15 (33.3 percent) of the samples were Bt cotton smuggled from Sudan. The PCR assay also revealed the presence of GM maize. Moreover, the effects of GM genes on the environment were studied in diseased samples, and no transgenes were found. Furthermore, domestic and indigenous crops were used to determine horizontal gene transfers of GM genes to other crops, and the transgene was not found in any of the samples analyzed. Conclusion: In the current study, 28 (13.4%) of the 209 (100%) total analyzed samples were GM crops which indicated the presence of unauthorized GM seeds in the study area. Environmental impact studies and horizontal gene transfer data similarly revealed that the Bt gene was not transferred to other crops and had no harmful environmental effects. For a better understanding of the Impact of imported unauthorized GM seeds, more additional detection of GM events should be done by expanding the sampling site and sample types.

First Report of Fusarium oxysporum f. sp. vasinfectum causing Fusarium Wilt of Cotton in Kansas, U.S.A.

Fusarium wilt is one of the most devastating diseases of cotton (Gossypium spp.). It is caused by the soil-borne pathogen Fusarium oxysporum Schlechtend. f. sp. vasinfectum (Atk.) Snyd. & Hans (Atkinson, 1892). To date, eight races of F. oxysporum f. sp. vasinfectum have been reported worldwide based on their diverse genetics and reactions to the host (Cianchetta, et al. 2015). Fusarium wilt symptoms appear at all developing stages. The fungus enters through the roots and colonizes the vascular system, leading to discoloration and wilting (Davis et al. 2006). During a survey in the July 2021 growing season, cotton plants showing typical wilting symptoms, stem discoloration, and root rot (Fig. 1A and B) were observed in two cotton fields in Sumner County, Kansas. In order to confirm the causal agent, root tissues and lower parts of the stems were collected from 25 diseased cotton plants and cleaned as previously described by Larren et al, (2001). Small segments (1-2 cm) of roots and stems were incubated on potato dextrose agar (PDA) at room temperature. The isolates were purified with the single spore method and 23 of 25 isolates (92%) showed typical morphology of F. oxysporum, with a white-peach pigmentation as reported previously (Leslie and Summerell 2008). Two isolates, CK13B and CK18A, one from each of the two fields were selected, and genomic DNA was extracted with E.Z.N.A Fungal DNA Mini kit (Omega Bio-tek, Norcross, GA). Polymerase chain reaction (PCR) assays were performed as reported previously (Ortiz, et al. 2017) to amplify and sequence a portion of three nuclear genes: translation elongation factor (EF-1α), phosphate: H* symporter (PHO), and β-tubulin (BT). BLASTn analysis of CK13B sequences showed 93-98% identity with F. oxysporum f. sp. vasinfectum isolates originating from Australia, with 93.43% (1706/1826 pb) identity of EF-1α to isolate AuSeed14 (KT323873), 95.11% (1849/1944 bp) of PHO to isolate AuK24232 (KT323909), and 97.56% (1840/1886 bp) identity of BT to isolate AusSeed14 (KT323833). Sequences of CK13B for EF1-α, PHO, and BT genes were submitted to GenBank, accession no. of ON754247, ON754248, and ON754249, respectively. The CK18A isolate showed high identity with F. oxysporum f. sp. vasinfectum races 1, 2, and 6 lineage isolates (Ortiz et al., 2017), with 99.89% (1822/1824 bp) identity of the EF1-α gene to isolate CDR238 (KT323838) from Arkansas, 98.36% (1916/1948 bp) identity of the PHO gene to isolate CDR1131 (KT323887) from Louisiana and 98.78% (1864/1887 bp) identity of the BT gene to isolate ATCC36198 (KT323799) from Brazil. Sequences of the CK18A isolate were submitted to GenBank, accession no. ON651444, ON725043, and ON725044, respectively. Pathogenicity of both isolates was tested on G. hirsutum as previously described (Kim et al. 2005) with three replicates per isolate and a control treatment in a growth chamber. Briefly, cotton seedlings were maintained with 12 h light/dark cycle at 25-20 °C. At the first true leaf stage seedlings were uprooted, roots were cleaned by rinsing with sterile water then placed in a conidial suspension (1×106 conidia/ml) for 2 h while the healthy control seedlings were dipped in water only. Plants were put into sterile soil, and the results were observed and recorded every seven days for a period of 30 days. The inoculated plants showed the same symptoms as those observed in the fields, wilting, leaf chlorosis, and necrosis. Both isolates were re-isolated from the roots of the inoculated plants. DNA was extracted and used for PCR with the specific gene primers as listed above. The sequences matched those of the original isolates, completing the Koch's pustulates. To our knowledge, this is the first report of F. oxysporum f. sp. vasinfectum causing Fusarium wilt disease of cotton in Kansas. These results will help growers select cultivars and design disease control strategies accordingly (Davis, et al., 2006). Further work is needed to determine the specific races in the area.

Construction and functional analysis of nattokinase-producing cucumber obtained by the CRISPR-Cas9 system

AbstractNattokinase (NK) is effective in the prevention and treatment of cardiovascular disease. Cucumber is rich in nutrients with low sugar content and is safe for consumption. The aim of this study was to construct a therapeutic cucumber that can express NK, which can prevent and alleviate cardiovascular diseases by consumption. Because the Bitter fruit (Bt) gene contributes to bitter taste but has no obvious effect on the growth and development of cucumber, so the NK-producing cucumber was constructed by replacing the Bt gene with NK by using CRISPR/Cas9. The pZHY988-Cas9-sgRNA and pX6-LHA-U6-NK-T-RHA vectors were constructed and transformed into Agrobacterium tumefaciens EHA105, which was transformed into cucumber by floral dip method. The crude extract of NK-producing cucumber had significant thrombolytic activity in vitro. In addition, treatment with the crude extract significantly delayed thrombus tail appearance, and the thrombin time of mice was much longer than that of normal mice. The degrees of coagulation and blood viscosity as well as hemorheological properties improved significantly after crude extract treatment. These findings show that NK-producing cucumber can effectively alleviate thrombosis and improve blood biochemical parameters, providing a new direction for diet therapy against cardiovascular diseases.

Development and utilization of analytical methods for rapid GM detection in processed food products: a case study for regulatory requirement

Genetically modified (GM) food crops for desired traits have been approved in some of the countries. The approval status of a GM event varies from country to country. In India, Bt cotton is the only approved GM crop. So far, no GM food crop has got regulatory approval in the country, which may be considered as unauthorized GM (UGM) event in the Indian context. The entry of UGM events of food crops needs to be checked in the imported consignments as well as in the marketplace. In the present study, screening elements were identified based on the genetically modified organism (GMO) matrix developed as decision support system for 22 GM food crops approved globally. Three sets of multiplex PCR assays were developed and validated for GM detection in food crops and products: triplex PCR targeting control elements [Cauliflower Mosaic Virus 35S promoter (P-35S), Figwort Mosaic Virus promoter (P-FMV), nopaline synthase terminator (T-nos)], triplex PCR targeting marker genes [aminoglycoside-3’-adenyltransferase (aadA), neomycin phosphotransferase (nptII), phosphinothricin-N-acetyltransferase (pat)] and duplex PCR targeting Bt genes (cry1Ab/Ac and cry2Ab2). Limit of detection (LOD) ranged from 0.5–0.01% for different targets, which is in compliance with the labelling threshold of many countries. The developed assays were utilized to check the GM status of apple and maize products along with an additional test for ctp2-cp4epsps for herbicide tolerance in maize. Based on the tests conducted, none of these samples were found positive for GM targets screened. These procedures could be efficiently employed as a part of GMO testing to trace GM contamination, if any, in both the imported as well as domestic food products.

Effect of Bt traits on transgenic rice’s growth and weed competitiveness

Transgene escape could lead to genetically modified rice establishing wild populations in the natural environment and competing for survival space with weeds. However, whether the expression of the Bacillus thuringiensis (Bt) gene in rice will alter the relationship between transgene plants and weeds and induce undesirable environmental consequences are poorly understood. Thus, field experiments were conducted to investigate the weed competitiveness and assess the ecological risk of transgenic Bt rice under herbicide-free and lepidopterous pest-controlled environments. Results showed that weed–rice competition in the direct-sowing (DS) field was earlier and more severe than that in the transplanting (TP) field, which resulted in a significant decrease in biomass and yield in DS. However, conventional Bt and non-Bt rice yield was not significantly different. The weed number, weed coverage ratio, and weed diversity of conventional Bt rice were significantly higher than those of non-Bt rice at the early growth and mature stages, especially in DS plots, suggesting that Bt traits did not increase the weed competitiveness of transgenic rice and had no negative effect on weed diversity. Grain yield and weed number varied between different hybrid rice lines, but those differences were insignificant between Bt and non-Bt rice. The number of insects increased with the increase of weeds in hybrid rice plots, whereas the insect number and diversity did not display a significant difference between Bt and non-Bt rice. Therefore, the ecological risk of transgenic Bt rice is comparable to non-Bt rice.

Impact of nitrogen and phosphorus fertilizers on Cry1Ac protein contents in transgenic cotton.

Application of different fertilizers to check the efficiency of expression of Bt (Bacillus thuringiensis) gene in one of the leading commercialized crops (cotton) against Lepidopteran species is of great concern. The expression of Cry protein level can be controlled by the improvement of nutrients levels. Therefore, the myth of response of Cry toxin to different combinations of NP fertilizers was explored in three Bt cotton cultivars. Combinations include three levels of nitrogen and three levels of phosphorus fertilizers. Immunostrips and Cry gene(s) specific primer based PCR (Polymerase Chain Reaction) analysis were used for the presence of Bt gene that unveiled the presence of Cry1Ac gene only. Further, the ELISA (enzyme-linked immunosorbent assay) kit was used to quantify the expression of Cry1Ac protein. Under various NP fertilizers rates, the level of toxin protein exhibited highly significant differences. The highest toxin level mean was found to be 2.3740 and 2.1732 µg/g under the treatment of N150P75 kg ha-1 combination while the lowest toxin level mean was found to be 0.9158 and 0.7641 µg/g at the N50P25 kg ha-1 level at 80 and 120 DAS (Days After Sowing), respectively. It was concluded from the research that the usage of NP fertilizers has a positive relation with the expression of Cry1Ac toxin in Bt cotton. We recommend using the N150P50 kg ha-1 level as the most economical and practicable fertilizer instead of the standard dose N100P50 kg ha-1 to get the desired level of Cry1Ac level for long lasting plant resistance (<1.5). The revised dose of fertilizer may help farmers to avoid the cross-resistance development in contradiction of insect pests.

Duplicate mutations of GhCYP450 lead to the production of ms5m6 male sterile line in cotton.

The duplicated male sterile genes ms5m6 in cotton were map-based cloned and validated by the virus-induced gene silencing assays. Duplicate mutations of the GhCYP450 gene encoding a cytochrome P450 protein are responsible for the male sterility in cotton. The utilization of male sterility in cotton plays a vital role in improving yield and fiber quality. A complete male sterile line (ms5ms6) has been extensively used to develop hybrid cotton worldwide. Using Zhongkang-A (ZK-A) developed by transferring Bt and ms5ms6 genes into the commercial cultivar Zhongmiansuo 12, the duplicate genes were map-based cloned and confirmed via the virus-induced gene silencing (VIGS) assays. The duplicate mutations of GhCYP450 genes encoding a cytochrome P450 protein were responsible for producing male sterility in ms5ms6 in cotton. Sequence alignment showed that GhCYP450-Dt in ZK-A differed in two critical aspects from the fertile wild-type TM-1: GhCYP450-Dt has three amino acid (D98E, E168K, G198R) changes in the coding region and a 7-bp (GGAAAAA) insertion in the promoter domain; GhCYP450-At appears to be premature termination of GhCYP450 translation. Further morphological observation and cytological examination of GhCYP450-silenced plants induced by VIGS exhibited shorter filaments and no mature pollen grains. These results indicate that GhCYP450 is essential for pollen exine formation and pollen development for male fertility. Investigating the mechanisms of ms5ms6 male sterility will deepen our understanding of the development and utilization of heterosis.

RAPD-markers assisted genetic diversity analysis and Bt-Cry1Ac gene identification in eggplant (Solanum melongena L.)

Generic diversity (GD) screening helps to identify candidate genes involved in diseases, pests, or other stress resistance in plants. GD is crucial for maintaining vigor and healthy plant population. The random amplified polymorphic DNA (RAPD) is a rapid polymerase chain reaction (PCR)-based molecular marker, effective for screening genetic diversity in diverse plant species. In this study, we applied the RAPD-markers-assisted PCR approach to explore genetic diversity (GD) among 7 varieties of eggplant (Solanum melongena L.) at DNA level. Further, we assessed the presence of Bt-Cry1Ac gene in those varieties using the PCR approach. The eggplants showed genetic diversity at the DNA level. The amplification with two RAPD primers (OPA-02, OPA-04) produced a total of 41 bands. The highest similarity showed 78.57% among sample 4 (Eggplant China) vs sample 7 (Purple king F1) and sample 1 (Eggplant Srilanka) vs sample 4 (Eggplant China), while the lowest was 28.57% among sample 7 (Purple king F1) vs sample 3 (Eggplant Chittagong-1). In addition, the PCR results for the Bt Cry1Ac primer did not show a considerable band in the expected amplicon size. The dendrogram showed the genetic variability and relationship among all seven eggplant varieties at the genetic level. The study formulated a simple marker-based method for an effective molecular protocol to study genetics in eggplants. It also reported the presence of the Bt gene in popular eggplant varieties.

Development and evaluation of triple gene transgenic cotton lines expressing three genes (Cry1Ac-Cry2Ab-EPSPS) for lepidopteran insect pests and herbicide tolerance

Cotton is an international agricultural commodity and the main cash crop of Pakistan of which quality and quantity are subject to various whims of nature. Climate change, insect pest complex, and weeds are reducing its productivity. Here, we have developed triple gene cotton containing EPSPS gene along with two Bt toxin genes Cry1Ac and Cry2Ab using a strategy where all three genes are cloned in the same T-DNA, followed by successful cotton transformation via Agrobacterium-mediated transformation. This strategy has been developed to help cotton breeders in developing new cultivars by incorporating these genes into the non-transgenic or single Bt (Cry1Ac) gene cotton background where all three genes will inherit together. The expression of all three proteins was confirmed through immunostrips and was quantified through enzyme-linked immunosorbent assay (ELISA). The spatio-temporal expression of Bt protein in different parts of triple gene NIBGE cotton plants was determined. Maximum expression was found in leaves followed by seeds and boll rinds. Insect bioassays with cotton bollworms (Helicoverpa armigera), armyworms (Spodoptera litura), and pink bollworms (Pectinophora gossypiella) showed more than 90% mortality. The best performing line (NIBGE-E2) on the basis of spatiotemporal expression, glyphosate assays, and insect mortality data, was used for event characterization by using the genome sequencing approach. The event was successfully characterized and named NIBGE 20-01. A diagnostics test based on event-specific PCR was developed and its ability to distinguish NIBGE 20-01 event from other commercial transgenic cotton events was confirmed. To confirm stable expression of all three proteins in the field conditions, homozygous transgenic lines were grown in the field and the expression was confirmed through immunostrip assays. It was found that all three genes are expressed under field conditions. To show that all three genes are inherited together upon crossing with local elite cotton lines, the F1 generation was grown under glasshouse and field conditions. The expression of all three genes was confirmed under field conditions. Our results showed that transgenic cotton with three genes cloned in the same T-DNA can express all genes and can be conveniently transferred into elite cotton lines through a single cross.

Introduction of Bacillus thuringiensis (Bt) gene does not reduce potassium use efficiency of Bt transgenic cotton (Gossypium hirsutum L.)

BackgroundPotassium (K) deficiency has become a common field production problem following the widespread adoption of Bacillus thuringiensis (Bt) transgenic cotton (Gossypium hirsutum L.) worldwide. The purpose of this study was to clarify whether the introduction of Bt gene directly reduces the K-use efficiency of cotton to induce K deficiency.ResultsThe cotton variety, Jihe 321 (wild type, WT) and its two Bt (Cry1Ac)-transgenic overexpression lines (OE-29317, OE-29312) were studied in field with low soil-test K+ (47.8 mg·kg−1). In the field with low soil-test K+, only OE-29317 had less biomass and K+ accumulation than the WT at some growth stages. Both Bt lines produced similar or even greater seed cotton yield than WT in the field. When the Bt gene (~ 70%) in OE-29317 and OE-29312 plants was silenced by virus-induced gene silencing (VIGS), the VIGS-Bt plants did not produce more biomass than VIGS-green fluorescent protein (control) plants.ConclusionsThe introduction of Bt gene did not necessarily hinder the K use efficiency of the cotton lines under this study.

P317 Phaeohyphomycosis caused by Phialophora verrucosa in a patient with CARD9 mutations

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PMObjectivesTo study an intractable case of phaeohyphomycosis caused by Phialophora verrucosa and the underlying genetic and immunological mechanisms.MethodsClinical and laboratory findings of the patient were studied. The patient's DNA was sequenced for CARD9. Innate and adaptive immunological responses of patient-derived PBMCs were evaluated with ELISA and flow cytometry.ResultsA 29-year-old Chinese male, born to nonconsanguineous parents, developed an enlarging plaque on his right anterior tibia at 25 years and underwent a 15-month treatment of oral itraconazole at the local hospital. Upon discontinuation of the medication, the plaque spread over his face, upper limbs, and back. Physical examination indicated dark brown verrucous plaques and nodules with surface ulcers and scabs on his right leg, upper limbs, and face. Skin biopsy revealed pigmented yeast cells with budding and yeast cell chain in the dermis. Based on tissue culture, we identified P. verrucosa as the causative pathogen, which was further validated by ITS, BT, and TEF gene sequencing. Thus, subcutaneous phaeohyphomycosis was diagnosed, and itraconazole combined with amphotericin B was prescribed. The lesions improved only marginally. We, therefore, gave him oral posaconazole instead, based on in vitro antifungal susceptibility results. After 1 month of posaconazole treatment, the serum BG assay value decreased and the lesions improved markedly.Based on previous reports linking CARD9 deficiency to phaeohyphomycosis, as well as early onset and recalcitrance characters, Sanger sequencing was performed in this patient and identified two compound heterozygous mutations in exon 6 (R373P) and exon 8 (D274GfsX60) of CARD9. We then evaluated the functional effect of the CARD9 mutations by assessing the stimuli-dependent release of proinflammatory cytokines and helper T cell differentiation. Cytokine production was markedly impaired in PBMCs post 24 h of stimulation with LPS, curdlan, TDB, mannan, MDP, β‐glucan, and various fungal ligands. Moreover, the patient showed a significant absence in Th22 and Th17 cell proportions, as well as lower IL-17A and IL-22 production after six days of co-incubation with the stimuli mentioned above.ConclusionOur report highlighted that otherwise healthy patients diagnosed with early-onset, unexplained, and recalcitrant phaeohyphomycosis should be analyzed for CARD9 mutations and immune deficiency. Furthermore, posaconazole may be an alternative worth trying after the failure of other antifungals due to its ideal results achieved in our case.

Effects of Exogenous Salicylic Acid Application to Aboveground Part on the Defense Responses in Bt (Bacillus thuringiensis) and Non-Bt Corn (Zea mays L.) Seedlings.

Bt (Bacillus thuringiensis) corn is one of the top three large-scale commercialized anti-insect transgenic crops around the world. In the present study, we tested the Bt protein content, defense chemicals contents, and defense enzyme activities in both the leaves and roots of Bt corn varieties 5422Bt1 and 5422CBCL, as well as their conventional corn 5422 seedlings, with two fully expanded leaves which had been treated with 2.5 mM exogenous salicylic acid (SA) to the aboveground part for 24 h. The result showed that the SA treatment to the aboveground part could significantly increase the polyphenol oxidase activity of conventional corn 5422, the Bt protein content, and peroxidase activities of Bt corn 5422Bt1, as well as the polyphenol oxidase and peroxidase activity of Bt corn 5422CBCL in the leaves. In the roots, the polyphenol oxidase and peroxidase activity of conventional corn 5422, the polyphenol oxidase and superoxide dismutase activities of Bt corn 5422Bt1, the DIMBOA (2,4-dihydroxy-7-methoxy-2H, 1, 4-benzoxazin-3 (4H)-one) content, and four defense enzymes activities of Bt corn 5422CBCL were systematically increased. These findings suggest that the direct effect of SA application to aboveground part on the leaf defense responses in Bt corn 5422CBCL is stronger than that in non-Bt corn. Meanwhile, the systemic effect of SA on the root defense responses in Bt corn 5422CBCL is stronger than that in conventional corn 5422 and Bt corn 5422Bt1. It can be concluded that the Bt gene introduction and endogenous chemical defense responses of corns act synergistically during the SA-induced defense processes to the aboveground part. Different transformation events affected the root defense response when the SA treatment was applied to the aboveground part.

Resistance in Pink Bollworm Pectinophora gossypiella (Saunders) against Bt Cotton, a Major Threat to Cotton in India: A Brief Review

The pink bollworm, Pectinophora gossypiella (Lepidoptera: Gelechiidae) is a primary pest of cotton in many regions of the world. In many parts of India, pink bollworm (PBW) has become resistant to BG-I (Bollgard I®) that expresses a single Bt gene (Cry1Ac) in 2009 and BG-II (Cry1Ac and cry 2Ab) in 2015. The brief review addresses the possible reasons and solutions to resistance posed by this insect. The review addresses the following points: reasons for resistance in PBW is discussed; the life-history is discussed how it’s distinctness from other bollworms; the introduction of hybrid cotton lead to insecticide treadmill in India; reduced accomplishment of the refugia recommendations also contributed to the problem; poor compliance of the bio-safety laws also led to the problem; unfavorable crop management practices prove to be an add-on to the problem. Furthermore, the possible solutions of the problem are discussed; monitoring and mating disruption; cultivation of High-density short season (HD-SS) pure line varieties; case studies of sterile insect technique (SIT) proved to be supporting in control of PBW; RNA interference technique can overcome the problem of resistance; use of chemical insecticides in different manner can change the perspective; proper cultural or farming practices can make the difference; Bio-control agents can overcome the problem; extension functionaries can help in solving the PBW problem. Therefore the management of resistance in PBW requires a broader vision and development of technology.

Standardization of an efficient protocol for transformation in Indian cauliflower (Brassica oleracea) var. botrytis

Cauliflower is an important Cole crop grown all over the world for its nutritional benefits. But it is encountered by a serious problem caused by the insect- pest attack. Plutella xylostella is one such insect which causes huge losses. An efficient and simple method for genetic transformation and regeneration of Indian cauliflower, Brassica oleracea var. botrytis has to be developed. Hence, optimization of the plant transformation is a pre- requisite for successful transformation process. Therefore, we worked on this objective at the Indian Agricultural Research Institute, New Delhi, India, and optimized the Agrobacterium-mediated transformation protocol in Indian cauliflower, Pusa Snowball K1 and Pusa Snowball KT25 varieties for developing insect resistance against Plutella xylostella using Bt genes. The Agrobacterium tumefaciens mediated transformation process was utilized for carrying out the transformation and the explant was obtained from the seedlings germinated from the seeds of the respective variety and used for genetic transformation process. The transgene integration and expression were later confirmed by PCR and RT-qPCR, respectively. Moreover, insect bioassay showed that transgenic plants effectively controlled the Diamond Back moth infestation concerning to the control plants indicating the presence of resistance developed against the insect. Hence, this protocol efficiently regenerated transgenic plants that lead to the resistance against biotic stress, specific to the insect Diamond back moth.

Modeling fall armyworm resistance in Bt-maize areas during crop and off-seasons

Entomologists have often used computational modeling to study the dynamics of insects in agricultural landscapes. Recently, important issues such as the movement of adults and immatures associated with insect resistance to GMO (genetically modified organism) crops have been addressed using computational models. We developed an individual-based model using the cellular automata approach (CA) to investigate how an intercropping system composed of maize engineered with Bacillus thuringiensis (Bt) gene, refuge areas (non-Bt maize), and grasses combined with off-season periods might influence the evolution of resistance in Spodoptera frugiperda (Lepidoptera: Noctuidae), one of the leading agricultural pests targeted by GMOs. We designed the Bt and non-Bt plants in two different arrangements: (a) a seed mixture and (b) strips rows, adding grasses in areas adjacent to the field. We added the seasonal planting dynamics (crop season and off-season), to evaluate a total of six agricultural scenarios. We followed a crop calendar from the United States to create simulations close to agricultural practice. The results showed that the frequency of the resistance allele was strongly related to the landscape arrangements and their dynamics. Since the adult insects are mobile, the seed-mixture scenario increased the frequency of the resistance the most (95.86%), followed by strips (82.10%), without grass fields. The maize harvest made it possible to reduce the frequency of resistance allele below 1%. Based on our results, we can expect that the maintenance of pasture areas, for instance next to the corn crops, will act as a reservoir of susceptible insects during off-season periods.

Effect of the percentage of Bos taurus inheritance on the fertility of Holstein×Zebu and Brown Swiss×Zebu crossbred cows in the Mexican tropics

Background: No dairy breed or crossbreed has superior overall performance in all environments; therefore, it is necessary to determine which crossbreed is the most suitable for the Mexican tropic and what proportion of European breed is optimum for reproduction. Objective: To assess the effect of the proportion of Bos taurus (Bt) genes on reproductive performance of Holstein×Zebu (HZ) and Brown Swiss×Zebu (BZ) cows, and compare reproductive performance of these genotypes in a dual-purpose production system. Methods: Cows were maintained in a rotational grazing system on African star grass (Cynodon plectostachyus) in Veracruz, Mexico. Cows were milked twice daily. Calves were kept tied to the side of their dams while the cows were milked. Results: The percentage of Bt genes did not affect (p&gt;0.05) fertility traits (age at first calving, days to first service after calving, services per conception, conception rate at first service, days open until conception, gestation length, and calving interval) of BZ cows. In contrast, HZ cows with less than 75% Holstein (H) genes were 0.3 years younger (p&lt;0.05) at first calving and had 39.8 fewer days open (p&lt;0.05) than HZ cows with 75% H genes or more. In addition, the calving interval of HZ cows with less than 75% H genes was 44.8 days shorter (p&lt;0.05) than that of HZ cows with 75% H genes or more. The HZ cows had five fewer days pregnant and were 22.8 kg heavier at calving (p&lt;0.05) than BZ cows. Conclusions: The effect of the percentage of Bt genes on cow fertility depends on the dairy breed used. In general, BZ and HZ cows present similar reproductive performance.