Co-expression of high-molecular-weight glutenin subunit 1Ax1 and Puroindoline a (Pina) genes in transgenic durum wheat (Triticum turgidum ssp. durum) improves milling and pasting quality.

BACKGROUND: Durum wheat is considered not suitable for making many food products that bread wheat can. This limitation is largely due to: (i) lack of grain-hardness controlling genes (Puroindoline a and b) and consequently extremely-hard kernel; (ii) lack of high- and low-molecular-weight glutenin subunit loci (Glu-D1 and Glu-D3) that contribute to gluten strength. To improve food processing quality of durum wheat, we stacked transgenic Pina and HMW-glutenin subunit 1Ax1 in durum wheat and developed lines with medium-hard kernel texture. RESULTS: Here, we demonstrated that co-expression of Pina + 1Ax1 in durum wheat did not affect the milling performance that was enhanced by Pina expression. While stacking of Pina + 1Ax1 led to increased flour yield, finer flour particles and decreased starch damage compared to the control lines. Interestingly, Pina and 1Ax1 co-expression showed synergistic effects on the pasting attribute peak viscosity. Moreover, Pina and 1Ax1 co-expression suggests that PINA impacts gluten aggregation via interaction with gluten protein matrix. CONCLUSIONS: The results herein may fill the gap of grain hardness between extremely-hard durum wheat and the soft kernel durum wheat, the latter of which has been developed recently. Our results may also serve as a proof of concept that stacking Puroindolines and other genes contributing to wheat end-use quality from the A and/or D genomes could improve the above-mentioned bottleneck traits of durum wheat and help to expand its culinary uses.

The genetic and molecular basis of crop height based on a rice model.

MAIN CONCLUSION: This review presents genetic and molecular basis of crop height using a rice crop model. Height is controlled by multiple genes with potential to be manipulated through breeding strategies to improve productivity. Height is an important factor affecting crop architecture, apical dominance, biomass, resistance to lodging, tolerance to crowding and mechanical harvesting. The impressive increase in wheat and rice yield during the 'green revolution' benefited from a combination of breeding for high-yielding dwarf varieties together with advances in agricultural mechanization, irrigation and agrochemical/fertilizer use. To maximize yield under irrigation and high fertilizer use, semi-dwarfing is optimal, whereas extreme dwarfing leads to decreased yield. Rice plant height is controlled by genes that lie in a complex regulatory network, mainly involved in the biosynthesis or signal transduction of phytohormones such as gibberellins, brassinosteroids and strigolactones. Additional dwarfing genes have been discovered that are involved in other pathways, some of which are uncharacterized. This review discusses our current understanding of the regulation of plant height using rice as a well-characterized model and highlights some of the most promising research that could lead to the development of new, high-yielding varieties. This knowledge underpins future work towards the genetic improvement of plant height in rice and other crops.

Overexpression of BraLTP2, a Lipid Transfer Protein of Brassica napus, Results in Increased Trichome Density and Altered Concentration of Secondary Metabolites.

Plant non-specific lipid transfer proteins (nsLTPs) belong to a large multigene family that possesses complex physiological functions. Trichomes are present on the aerial surfaces of most plants and include both glandular secretory hairs and non-glandular hairs. In this study, BraLTP2 was isolated from Brassica rapa (B. rapa) and its function was characterized in the important oilseed crop Brassica napus (B. napus). B. rapa lipid transfer protein 2 (BraLTP2) belongs to the little-known Y class of nsLTPs and encodes a predicted secretory protein. In ProBraLTP2::GUS (ß-glucuronidase) transgenic plants, strong GUS activity was observed in young leaves and roots, while low activity was observed in the anther. It is noteworthy that strong GUS activity was observed in trichomes of the first four leaves of 4-week-old and 8-week-old seedings, however, it disappeared in 12-week-old seedings. In transgenic plants expressing a BraLTP2::GFP (green fluorescent protein) fusion protein, GFP fluorescence localized in the extracellular space of epidermal cells and trichomes. Overexpression of BraLTP2 in B. napus caused an increase in trichome number and altered the accumulation of secondary metabolites in leaves, including 43 upregulated secondary metabolites. Moreover, transgenic plants showed significantly increased activities of antioxidant enzymes. These results suggest that BraLTP2, a new nsLTP gene, may play a role in trichome development and the accumulation of secondary metabolites.

A Review of Erucic Acid Production in Brassicaceae Oilseeds: Progress and Prospects for the Genetic Engineering of High and Low-Erucic Acid Rapeseeds (Brassica napus).

Erucic acid (C22:1, ω-9, EA) is a very-long-chain monounsaturated fatty acid (FA) that is an important oleochemical product with a wide range of uses in metallurgy, machinery, rubber, the chemical industry, and other fields because of its hydrophobicity and water resistance. EA is not easily digested and absorbed in the human body, and high-EA rapeseed (HEAR) oil often contains glucosinolates. Both glucosinolates and EA are detrimental to health and can lead to disease, which has resulted in strict guidelines by regulatory bodies on maximum EA contents in oils. Increasingly, researchers have attempted to enhance the EA content in Brassicaceae oilseeds to serve industrial applications while conversely reducing the EA content to ensure food safety. For the production of both LEAR and HEAR, biotechnology is likely to play a fundamental role. Elucidating the metabolic pathways of EA can help inform the improvement of Brassicaceae oilseeds through transgenic technology. In this paper, we introduce the industrial applications of HEAR oil and health benefits of low-EA rapeseed (LEAR) oil first, following which we review the biosynthetic pathways of EA, introduce the EA resources from plants, and focus on research related to the genetic engineering of EA in Brassicaceae oilseeds. In addition, the effects of the environment on EA production are addressed, and the safe cultivation of HEAR and LEAR is discussed. This paper supports further research into improving FAs in Brassicaceae oilseeds through transgenic technologies and molecular breeding techniques, thereby advancing the commercialization of transgenic products for better application in various fields.

A Review of Nervonic Acid Production in Plants: Prospects for the Genetic Engineering of High Nervonic Acid Cultivars Plants.

Nervonic acid (NA) is a very-long-chain monounsaturated fatty acid that plays crucial roles in brain development and has attracted widespread research interest. The markets encouraged the development of a refined, NA-enriched plant oil as feedstocks for the needed further studies of NA biological functions to the end commercial application. Plant seed oils offer a renewable and environmentally friendly source of NA, but their industrial production is presently hindered by various factors. This review focuses on the NA biosynthesis and assembly, NA resources from plants, and the genetic engineering of NA biosynthesis in oil crops, discusses the factors that affect NA production in genetically engineered oil crops, and provides prospects for the application of NA and prospective trends in the engineering of NA. This review emphasizes the progress made toward various NA-related topics and explores the limitations and trends, thereby providing integrated and comprehensive insight into the nature of NA production mechanisms during genetic engineering. Furthermore, this report supports further work involving the manipulation of NA production through transgenic technologies and molecular breeding for the enhancement of crop nutritional quality or creation of plant biochemical factories to produce NA for use in nutraceutical, pharmaceutical, and chemical industries.

Comparison of three Agrobacterium-mediated co-transformation methods for generating marker-free transgenic Brassica napus plants.

BACKGROUND: Generation of marker-free transgenic plants is very important to the regulatory permission and commercial release of transgenic crops. Co-transformation methods that enable the removal of selectable marker genes have been extensively used because they are simple and clean. Few comparisons are currently available between different strain/plasmid co-transformation systems, and also data are related to variation in co-transformation frequencies caused by other details of the vector design. RESULTS: In this study, we constructed three vector systems for the co-transformation of allotetraploid Brassica napus (B. napus) mediated by Agrobacterium tumefaciens and compared these co-transformation methods. We tested a mixed-strain system, in which a single T-DNA is harbored in two plasmids, as well as two "double T-DNA" vector systems, in which two independent T-DNAs are harbored in one plasmid in a tandem orientation or in an inverted orientation. As confirmed by the use of PCR analysis, test strips, and Southern blot, the average co-transformation frequencies from these systems ranged from 24 to 81% in T0 plants, with the highest frequency of 81% for 1:1 treatment of the mixed-strain system. These vector systems are valuable for generating marker-free transgenic B. napus plants, and marker-free plants were successfully obtained in the T1 generation from 50 to 77% of T0 transgenic lines using these systems, with the highest frequency of 77% for "double T-DNA" vector systems of pBID RT Enhanced. We further found that marker-free B. napus plants were more frequently encountered in the progeny of transgenic lines which has only one or two marker gene copies in the T0 generation. Two types of herbicide resistant transgenic B. napus plants, Bar + with phosphinothricin resistance and Bar + EPSPS + GOX + with phosphinothricin and glyphosate resistance, were obtained. CONCLUSION: We were successful in removing selectable marker genes in transgenic B. napus plants using all three co-transformation systems developed in this study. It was proved that if a appropriate mole ratio was designed for the specific length ratio of the twin T-DNAs for the mixed-strain method, high unlinked co-insertion frequency and overall success frequency could be achieved. Our study provides useful information for the construction of efficient co-transformation system for marker-free transgenic crop production and developed transgenic B. napus with various types of herbicide resistance.

Expression of Puroindoline a in Durum Wheat Affects Milling and Pasting Properties.

Durum wheat has limited culinary utilizations partly due to its extremely hard kernel texture. Previously, we developed transgenic durum wheat lines with expression of the wildtype Puroindoline a (Pina) and characterized PINA's effects on kernel hardness, total flour yield and dough mixing properties in durum wheat. The medium-hard kernel texture is potentially useful for exploring culinary applications of durum wheat. In the present study, we examined the milling parameters and flour attributes of the transgenic lines, including particle size distribution, damaged starch and water binding capacity. PINA expression results in increased break and reduction flour yield but decreased shorts. PINA expression also leads to finer flour particles and decreased starch damage. Interestingly, PINA transgenic lines showed increased peak viscosity and breakdown viscosity but leave other flour pasting parameters generally unaltered. PINA transgenic lines were associated with increased small monomeric proteins, appearing to affect gluten aggregation. Our data together with several previous results highlight distinct effects of PINs on pasting properties depending on species and variety. The medium-hard kernel texture together with improved pasting parameters may be valuable for producing a broader range of end-products from durum wheat.