Convergence of Bar and Cry1Ac Mutant Genes in Soybean Confers Synergistic Resistance to Herbicide and Lepidopteran Insects

Soybean is a globally important crop species, which is subject to pressure by insects and weeds causing severe substantially reduce yield and quality. Despite the success of transgenic soybean in terms of Bacillus thuringiensis (Bt) and herbicide tolerance, unforeseen mitigated performances have still been inspected due to climate changes that favor the emergence of insect resistance. Therefore, there is a need to develop a biotech soybean with elaborated gene stacking to improve insect and herbicide tolerance in the field. In this study, new gene stacking soybean events, such as bialaphos resistance (bar) and pesticidal crystal protein (cry)1Ac mutant 2 (M#2), are being developed in Vietnamese soybean under field condition. Five transgenic plants were extensively studied in the herbicide effects, gene expression patterns, and insect mortality across generations. The increase in the expression of the bar gene by 100% in the leaves of putative transgenic plants was a determinant of herbicide tolerance. In an insect bioassay, the cry1Ac-M#2 protein tested yielded higher than expected larval mortality (86%), reflecting larval weight gain and weight of leaf consumed were less in the T1 generation. Similarly, in the field tests, the expression of cry1Ac-M#2 in the transgenic soybean lines was relatively stable from T0 to T3 generations that corresponded to a large reduction in the rate of leaves and pods damage caused by Lamprosema indicata and Helicoverpa armigera. The transgenic lines converged two genes, producing a soybean phenotype that was resistant to herbicide and lepidopteran insects. Furthermore, the expression of cry1Ac-M#2 was dominant in the T1 generation leading to the exhibit of better phenotypic traits. These results underscored the great potential of combining bar and cry1Ac mutation genes in transgenic soybean as pursuant of ensuring resistance to herbicide and lepidopteran insects.

Gene stacking: Approach of genetic engineering

Gene stacking is the process of addition of two or more gene of interest into a single plant. The combination or stacking of different traits or genes in plants is rapidly gaining popularity in biotech crop production. The new evolved trait is known as stacked trait and the crop is known as biotech stacked or simply stacked. This can be accomplished in many ways, one of which is gene pyramiding. Biotech stacks give crops a larger genetic and agronomic boost, allowing them to perform better in challenging farming situations. Biotech stacks are designed to increase productivity by overcoming biotic and abiotic challenges like as insect pests, diseases, weeds, and environmental stress. This review will explain about the gene stacking principle, the need for biotech stacking, and the many gene stacking methods.

Molecular marker assisted gene stacking for disease resistance and quality genes in the dwarf mutant of an elite common wheat cultivar Xiaoyan22

Background: Development of wheat cultivars with multiple disease resistance and high quality are major objectives in modern wheat breeding programs. Gene stacking is an efficient approach to achieve this target. In this study, we pyramided yellow rust resistance gene (Yr26), powdery mildew resistance gene (ML91260) and high-molecular-weight glutenin subunits Dx5+Dy10 into the dwarf mutant of an elite wheat cultivar, Xiaoyan22. Results: Six pyramided wheat lines were obtained by molecular marker-assisted selection (MAS) and field evaluation of disease resistance. The desirable agronomic traits of pyramided lines, their identity with the original cultivar Xiaoyan22 except for plant height, tiller number and disease resistance, was achieved in this study. Meanwhile, the yield of pyramided lines is higher than Xiaoyan22 in the field test. In addition, analysis of flour quality indicated that the dough stability time of pyramided lines was longer than that of Xiaoyan22. Conclusions: Six pyramided wheat lines with two disease resistance and high quality were achieved in this study. It is feasible to improve multiple agronomic traits simultaneously by rational application of MAS.

Gene stacking as a strategy to confer characteristics of agronomic importance in plants by genetic engineering

ABSTRACT: Gene stacking refers to the introduction of two or more transgenes of agronomic interest in the same plant. The main methods for genetically engineering plants with gene stacking involve (i) the simultaneous introduction, by the co-transformation process, and (ii) the sequential introduction of genes using the re-transformation processes or the sexual crossing between separate transgenic events. In general, the choice of the best method varies according to the species of interest and the availability of genetic constructions and preexisting transgenic events. We also present here the use of minichromosome technology as a potential future gene stacking technology. The purpose of this review was to discuss aspects related to the methodology for gene stacking and trait stacking (a gene stacking strategy to combine characteristics of agronomical importance) by genetic engineering. In addition, we presented a list of crops and genes approved commercially that have been used in stacking strategies for combined characteristics and a discussion about the regulatory standards. An increased number of approved and released gene stacking events reached the market in the last decade. Initially, the most common combined characteristics were herbicide tolerance and insect resistance in soybean and maize. Recently, commercially available varieties were released combining these traits with drought tolerance in these commodities. New traits combinations are reaching the farmer’s fields, including higher quality, disease resistant and nutritional value improved. In other words, gene stacking is growing as a strategy to contribute to food safety and sustainability.

Molecular marker assisted gene stacking for disease resistance and quality genes in the dwarf mutant of an elite common wheat cultivar Xiaoyan22

Development of wheat cultivars with multiple disease resistance and high quality are major objectives in modern wheat breeding programs. Gene stacking is an efficient approach to achieve this target. In this study, we pyramided yellow rust resistance gene (Yr26), powdery mildew resistance gene (ML91260) and high-molecular-weight glutenin subunits Dx5+Dy10 into the dwarf mutant of an elite wheat cultivar, Xiaoyan22. Six pyramided lines were obtained by molecular marker-assisted selection (MAS) and field evaluation of disease resistance. The desirable agronomic traits of pyramided lines, their identity with the original cultivar Xiaoyan22 except for plant height, tiller number and disease resistance, was achieved in this study. Meanwhile, the yield of pyramided lines is higher than Xiaoyan22 in the field test. In addition, analysis of flour quality indicated that the dough stability time of pyramided lines was longer than that of Xiaoyan22. These results demonstrate that it is feasible to improve multiple agronomic traits simultaneously by rational application of MAS.

Development of an amenable system for site-specific addition to a maize chromosome

Currently, transgenic maize is produced by random integration of a transgenes into the plant. This works for single genes, but not as well for multiple traits. Identifying plants that contain several transgenes becomes a very difficult task. Gene stacking at a single location in the genome would make combining multiple transgenes into plants a simpler process. This project focused on the development of a system would allow for transgenes to be sequentially added to a specific site in the maize genome. The system utilizes two recombinases, Cre recombinase and _C31 Integrase, to remove a selectable marker and to integrate transgenes. An initial construct containing a selectable marker, flanked by LoxP sites, which are acted upon by Cre recombinase, and an attP site, were transformed. The selectable marker was then removed from the integrated transgene by exposure to Cre recombinase. Two amendment constructs enable modification of the integrated construct by utilizing complementary attP and attB sites, which are acted upon by _C31 Integrase. The amendment constructs contain cargo and a promoterless selectable marker which, upon successful recombination with the target site, restores expression of the selectable marker. Successful demonstration of this system simplifies generation of multi-transgene plants, and the assembly of multi-gene pathways in plants.

Molecular marker assisted gene stacking for disease resistance and quality genes in the dwarf mutant of an elite wheat cultivar Xiaoyan22

Development of wheat cultivars with multiple disease resistance and high quality are major objectives in modern wheat breeding programs. Gene stacking is an efficient approach to achieve this target. In this study, we pyramided yellow rust resistance gene (Yr26), powdery mildew resistance gene (ML91260) and high-molecular-weight glutenin subunits Dx5+Dy10 into the dwarf mutant of an elite wheat cultivar, Xiaoyan22. Six pyramided lines were obtained by molecular marker-assisted selection (MAS) and field evaluation of disease resistance. The desirable agronomic traits of pyramided lines, their identity with the original cultivar Xiaoyan22 except for plant height, tiller number and disease resistance, was achieved in this study. Meanwhile, the yield of pyramided lines is higher than Xiaoyan22 in the field test. In addition, analysis of flour quality indicated that the dough stability time of pyramided lines was longer than that of Xiaoyan22. These results demonstrate that it is feasible to improve multiple agronomic traits simultaneously by rational application of MAS.