Simultaneous improvement of fiber yield and quality in upland cotton (Gossypium hirsutum L.) by integration of auxin transport and synthesis.

Cotton is a widely planted commercial crop in the world. Enhancing fiber yield and quality is a long-term goal for cotton breeders. Our previous work has demonstrated that fine promotion of auxin biosynthesis in ovule epidermis, by overexpressing FBP7pro::iaaM, has a significant improvement on lint yield and fiber fineness. Lately, transgenic cottons overexpressing GhROP6 variants modify mature fiber length by controlling GhPIN3a-mediated polar auxin transport in ovules. Here, this study showed that all these GhROP6-related cottons displayed unsatisfactory agronomic performance in field conditions. Yet extra auxin supply could promote their fiber development, suggesting inadequate auxin supply in the ovules. Thus, these cottons were integrated with enhanced auxin synthesis by crossing with FBP7pro::iaaM cotton. All the transgene-stacked cottons exhibited synergetic effects on cotton yield (seedcotton yield, lint yield, and lint percentage) and quality (length, strength, and micronaire). Notably, comparing to the FBP7pro::iaaM background, the transgene-stacked cotton co-expressing FBP7pro::iaaM and CA-ghrop6 (constitutively active GhROP6) exhibited a 12.6% increase in seedcotton yield and a 19.0% increase in lint yield over a three-year field trial, and simultaneously resulted in further improvement on fiber length, strength, and micronaire. Collectively, our data provide a potential strategy for genetic improvement on cotton fiber yield and quality. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01500-w.

Impact of Intron and Retransformation on Transgene Expression in Leaf and Fruit Tissues of Field-Grown Pear Trees.

Stable and high expression of introduced genes is a prerequisite for using transgenic trees. Transgene stacking enables combining several valuable traits, but repeated transformation increases the risk of unintended effects. This work studied the stability and intron-mediated enhancement of uidA gene expression in leaves and different anatomical parts of pear fruits during field trials over 14 years. The stability of reporter and herbicide resistance transgenes in retransformed pear plants, as well as possible unintended effects using high-throughput phenotyping tools, were also investigated. The activity of ß-glucuronidase (GUS) varied depending on the year, but silencing did not occur. The uidA gene was expressed to a maximum in seeds, slightly less in the peel and peduncles, and much less in the pulp of pear fruits. The intron in the uidA gene stably increased expression in leaves and fruits by approximately twofold. Retransformants with the bar gene showed long-term herbicide resistance and exhibited no consistent changes in leaf size and shape. The transgenic pear was used as rootstock and scion, but grafted plants showed no transport of the GUS protein through the graft in the greenhouse and field. This longest field trial of transgenic fruit trees demonstrates stable expression under varying environmental conditions, the expression-enhancing effect of intron and the absence of unintended effects in single- and double-transformed woody plants.

Anthocyanins in Plant Food: Current Status, Genetic Modification, and Future Perspectives.

Anthocyanins are naturally occurring polyphenolic pigments that give food varied colors. Because of their high antioxidant activities, the consumption of anthocyanins has been associated with the benefit of preventing various chronic diseases. However, due to natural evolution or human selection, anthocyanins are found only in certain species. Additionally, the insufficient levels of anthocyanins in the most common foods also limit the optimal benefits. To solve this problem, considerable work has been done on germplasm improvement of common species using novel gene editing or transgenic techniques. This review summarized the recent advances in the molecular mechanism of anthocyanin biosynthesis and focused on the progress in using the CRISPR/Cas gene editing or multigene overexpression methods to improve plant food anthocyanins content. In response to the concerns of genome modified food, the future trends in developing anthocyanin-enriched plant food by using novel transgene or marker-free genome modified technologies are discussed. We hope to provide new insights and ideas for better using natural products like anthocyanins to promote human health.

Assembling multiple xenoprotective transgenes in pigs.

This review gives a brief overview of the genetic modifications necessary for grafted porcine tissues and organs to overcome rejection in human recipients. It then focuses on the problem of generating and breeding herds of donor pigs carrying modified endogenous genes and multiple xenoprotective transgenes. A xenodonor pig optimised for human clinical use could well require the addition of ten or more xenoprotective transgenes. It is impractical to produce the required combination of transgene by cross-breeding animals bearing individual transgenes at unlinked genetic loci, because independent segregation means that huge numbers of pigs would be required to produce relatively few donor animals. A better approach is to colocate groups of transgenes at a single genomic locus. We outline current methods to assemble transgene arrays and consider their pros and cons. These include polycistronic expression systems, in vitro recombination of large DNA fragments in PAC and BAC vectors, transposon vectors, classical gene targeting by homologous recombination at permissive loci such as ROSA26, targeted transgene placement aided by gene editing systems such as CRISPR/Cas9, and transgene placement by site-specific recombination such as Min-tagging using the Bxb1recombinase.

Strong xenoprotective function by single-copy transgenes placed sequentially at a permissive locus.

BACKGROUND: Multiple xenoprotective transgenes are best grouped at a single locus to avoid segregation during breeding and simplify production of donor animals. METHODS: We used transgene stacking to place a human CD55 transgene adjacent to a human heme oxygenase 1 construct at the porcine ROSA26 locus. A transgenic pig was analyzed by PCR, RT-PCR, droplet digital PCR, immunohistochemistry, immunofluorescence, and flow cytometry. Resistance to complement-mediated cell lysis and caspase 3/7 activation were determined in vitro. RESULTS: The ROSA26 locus was retargeted efficiently, and animals were generated by nuclear transfer. RNA and protein analyses revealed abundant expression in all organs analyzed, including pancreatic beta cells. Transgenic porcine kidney fibroblasts were almost completely protected against complement-mediated lysis and showed reduced caspase 3/7 activation. CONCLUSION: Step-by-step placement enables highly expressed single-copy xenoprotective transgenes to be grouped at porcine ROSA26.

A trait stacking system via intra-genomic homologous recombination.

MAIN CONCLUSION: A gene targeting method has been developed, which allows the conversion of 'breeding stacks', containing unlinked transgenes into a 'molecular stack' and thereby circumventing the breeding challenges associated with transgene segregation. A gene targeting method has been developed for converting two unlinked trait loci into a single locus transgene stack. The method utilizes intra-genomic homologous recombination (IGHR) between stably integrated target and donor loci which share sequence homology and nuclease cleavage sites whereby the donor contains a promoterless herbicide resistance transgene. Upon crossing with a zinc finger nuclease (ZFN)-expressing plant, double-strand breaks (DSB) are created in both the stably integrated target and donor loci. DSBs flanking the donor locus result in intra-genomic mobilization of a promoterless selectable marker-containing donor sequence, which can be utilized as a template for homology-directed repair of a concomitant DSB at the target locus resulting in a functional selectable marker via nuclease-mediated cassette exchange (NMCE). The method was successfully demonstrated in maize using a glyphosate tolerance gene as a donor whereby up to 3.3 % of the resulting progeny embryos cultured on selection medium regenerated plants with the donor sequence integrated into the target locus. The process could be extended to multiple cycles of trait stacking by virtue of a unique intron sequence homology for NMCE between the target and the donor loci. This is the first report that describes NMCE via IGHR, thereby enabling trait stacking using conventional crossing.

Developing carotenoids-enhanced tomato fruit with multi-transgene stacking strategies.

As natural dominant pigments, carotenoids and their derivatives not only contribute to fruit color and flavor quality but are regarded as phytochemicals beneficial to human health because of various bioactivities. Tomato is one of the most important vegetables as well as a main dietary source of carotenoids. So, it's of great importance to generate carotenoid-biofortified tomatoes. The carotenoid biosynthesis pathway is a network co-regulated by multiple enzymes and regulatory genes. Here, we assembled four binary constructs containing different combinations of four endogenous carotenoids metabolic-related genes, including SlORHis, SlDXS, SlPSY, and SlBHY by using a high efficiency multi-transgene stacking system and a series of fruit-specific promotors. Transgenic lines overexpression SlORHis alone, three genes (SlORHis/SlDXS/SlPSY), two genes (SlORHis/SlBHY), and all these four genes (SlORHis/SlDXS/SlPSY/SlBHY) were enriched with carotenoids to varying degrees. Notably, overexpressing SlORHis alone showed comparable effects with simultaneous overexpression of the key regulatory enzyme coding genes SlDXS, SlPSY, and SlORHis in promoting carotenoid accumulation. Downstream carotenoid derivatives zeaxanthin and violaxanthin were detected only in lines containing SlBHY. In addition, the sugar content and total antioxidant capacity of these carotenoids-enhanced tomatoes was also increased. These data provided useful information for the future developing of biofortified tomatoes with different carotenoid profiles, and confirmed a promising system for generation of nutrients biofortified tomatoes by multiple engineering genes stacking strategy.

Trait stacking via targeted genome editing.

Modern agriculture demands crops carrying multiple traits. The current paradigm of randomly integrating and sorting independently segregating transgenes creates severe downstream breeding challenges. A versatile, generally applicable solution is hereby provided: the combination of high-efficiency targeted genome editing driven by engineered zinc finger nucleases (ZFNs) with modular 'trait landing pads' (TLPs) that allow 'mix-and-match', on-demand transgene integration and trait stacking in crop plants. We illustrate the utility of nuclease-driven TLP technology by applying it to the stacking of herbicide resistance traits. We first integrated into the maize genome an herbicide resistance gene, pat, flanked with a TLP (ZFN target sites and sequences homologous to incoming DNA) using WHISKERS™-mediated transformation of embryogenic suspension cultures. We established a method for targeted transgene integration based on microparticle bombardment of immature embryos and used it to deliver a second trait precisely into the TLP via cotransformation with a donor DNA containing a second herbicide resistance gene, aad1, flanked by sequences homologous to the integrated TLP along with a corresponding ZFN expression construct. Remarkably, up to 5% of the embryo-derived transgenic events integrated the aad1 transgene precisely at the TLP, that is, directly adjacent to the pat transgene. Importantly and consistent with the juxtaposition achieved via nuclease-driven TLP technology, both herbicide resistance traits cosegregated in subsequent generations, thereby demonstrating linkage of the two independently transformed transgenes. Because ZFN-mediated targeted transgene integration is becoming applicable across an increasing number of crop species, this work exemplifies a simple, facile and rapid approach to trait stacking.

An Efficient Modular Gateway Recombinase-Based Gene Stacking System for Generating Multi-Trait Transgenic Plants.

Transgenic technology can transfer favorable traits regardless of reproductive isolation and is an important method in plant synthetic biology and genetic improvement. Complex metabolic pathway modification and pyramiding breeding strategies often require the introduction of multiple genes at once, but the current vector assembly systems for constructing multigene expression cassettes are not completely satisfactory. In this study, a new in vitro gene stacking system, GuanNan Stacking (GNS), was developed. Through the introduction of Type IIS restriction enzyme-mediated Golden Gate cloning, GNS allows the modular, standardized assembly of target gene expression cassettes. Because of the introduction of Gateway recombination, GNS facilitates the cloning of superlarge transgene expression cassettes, allows multiple expression cassettes to be efficiently assembled in a binary vector simultaneously, and is compatible with the Cre enzyme-mediated marker deletion mechanism. The linked dual positive-negative marker selection strategy ensures the efficient acquisition of target recombinant plasmids without prokaryotic selection markers in the T-DNA region. The host-independent negative selection marker combined with the TAC backbone ensures the cloning and transfer of large T-DNAs (>100 kb). Using the GNS system, we constructed a binary vector containing five foreign gene expression cassettes and obtained transgenic rice carrying the target traits, proving that the method developed in this research is a powerful tool for plant metabolic engineering and compound trait transgenic breeding.

Transgene Stacking as Effective Tool for Enhanced Disease Resistance in Plants.

Introduction of more than one gene into crop plants simultaneously or sequentially, called transgene stacking, has been a more effective strategy for conferring higher and durable insect and disease resistance in transgenic plants than single-gene technology. Transgenes can be stacked against one or more pathogens or for traits such as herbicide tolerance or anthocyanin pigmentation. Polygenic agronomic traits can be improved by multiple gene transformation. The most widely engineered stacked traits are insect resistance and herbicide tolerance as these traits may lead to lesser use of pesticides, higher yield, and efficient control of weeds. In this review, we summarize transgene stacking of two or more transgenes into crops for different agronomic traits, potential applications of gene stacking, its limitations and future prospects.

Plant Synthetic Metabolic Engineering for Enhancing Crop Nutritional Quality.

Nutrient deficiencies in crops are a serious threat to human health, especially for populations in poor areas. To overcome this problem, the development of crops with nutrient-enhanced traits is imperative. Biofortification of crops to improve nutritional quality helps combat nutrient deficiencies by increasing the levels of specific nutrient components. Compared with agronomic practices and conventional plant breeding, plant metabolic engineering and synthetic biology strategies are more effective and accurate in synthesizing specific micronutrients, phytonutrients, and/or bioactive components in crops. In this review, we discuss recent progress in the field of plant synthetic metabolic engineering, specifically in terms of research strategies of multigene stacking tools and engineering complex metabolic pathways, with a focus on improving traits related to micronutrients, phytonutrients, and bioactive components. Advances and innovations in plant synthetic metabolic engineering would facilitate the development of nutrient-enriched crops to meet the nutritional needs of humans.

Co-expression of multiple heavy metal transporters changes the translocation, accumulation, and potential oxidative stress of Cd and Zn in rice (Oryza sativa).

The OsHMA2, OsLCT1 and OsZIP3 transporters were all involved in zinc (Zn) and cadmium (Cd) transport. So far, only a few researches studied on the co-regulation effect of three transporters related to Zn and Cd transport. The present study showed that rice co-expressing OsLCT1-OsHMA2-OsZIP3 (LHZ) had longer roots and shoots than wild-type (WT) rice after Zn and Cd treatments. The chlorophyll content was significantly higher, and the proline, malondialdehyde and H2O2 contents were significantly lower in co-transgenic lines than in WT under Cd and Zn stress. LHZ in the seedlings of transgenic rice decreased the root-to-shoot translocation of Cd after Cd and Zn treatments. At the filling stage, LHZ line reduced Cd accumulation in grain after Cd treatment. Moreover, LHZ line increased the translocation of Zn to grain and reduced the accumulation of Cd after Zn treatment. These results suggested that LHZ co-expression could effectively decrease the translocation and accumulation of Cd to grains, alleviated the oxidative stress of Cd and Zn, and finally enhanced the quality and safety of rice grains.

Transgene Pyramiding of Salt Responsive Protein 3-1 (SaSRP3-1) and SaVHAc1 From Spartina alterniflora L. Enhances Salt Tolerance in Rice.

The transgenic technology using a single gene has been widely used for crop improvement. But the transgenic pyramiding of multiple genes, a promising alternative especially for enhancing complexly inherited abiotic stress tolerance, has received little attention. Here, we developed and evaluated transgenic rice lines with a single Salt Responsive Protein 3-1 (SaSRP3-1) gene as well as pyramids with two-genes SaSRP3-1 and Vacuolar H+-ATPase subunit c1 (SaVHAc1) derived from a halophyte grass Spartina alterniflora L. for salt tolerance at seedling, vegetative, and reproductive stages. The overexpression of this novel gene SaSRP3-1 resulted in significantly better growth of E. coli with the recombinant plasmid under 600 mM NaCl stress condition compared with the control. During early seedling and vegetative stages, the single gene and pyramided transgenic rice plants showed enhanced tolerance to salt stress with minimal wilting and drying symptoms, improved shoot and root growth, and significantly higher chlorophyll content, relative water content, and K+/Na+ ratio than the control plants. The salt stress screening during reproductive stage revealed that the transgenic plants with single gene and pyramids had better grain filling, whereas the pyramided plants showed significantly higher grain yield and higher grain weight compared to control plants. Our study demonstrated transgenic pyramiding as a viable approach to achieve higher level of salt tolerance in crop plants.

Development of "Purple Endosperm Rice" by Engineering Anthocyanin Biosynthesis in the Endosperm with a High-Efficiency Transgene Stacking System.

Anthocyanins have high antioxidant activities, and engineering of anthocyanin biosynthesis in staple crops, such as rice (Oryza sativa L.), could provide health-promoting foods for improving human health. However, engineering metabolic pathways for biofortification remains difficult, and previous attempts to engineer anthocyanin production in rice endosperm failed because of the sophisticated genetic regulatory network of its biosynthetic pathway. In this study, we developed a high-efficiency vector system for transgene stacking and used it to engineer anthocyanin biosynthesis in rice endosperm. We made a construct containing eight anthocyanin-related genes (two regulatory genes from maize and six structural genes from Coleus) driven by the endosperm-specific promoters,plus a selectable marker and a gene for marker excision. Transformation of rice with this construct generated a novel biofortified germplasm "Purple Endosperm Rice" (called "Zijingmi" in Chinese), which has high anthocyanin contents and antioxidant activity in the endosperm. This anthocyanin production results from expression of the transgenes and the resulting activation (or enhancement) of expression of 13 endogenous anthocyanin biosynthesis genes that are silenced or expressed at low levels in wild-type rice endosperm. This study provides an efficient, versatile toolkit for transgene stacking and demonstrates its use for successful engineering of a sophisticated biological pathway, suggesting the potential utility of this toolkit for synthetic biology and improvement of agronomic traits in plants.

Gene targeting and transgene stacking using intra genomic homologous recombination in plants.

Modern agriculture has created a demand for plant biotechnology products that provide durable resistance to insect pests, tolerance of herbicide applications for weed control, and agronomic traits tailored for specific geographies. These transgenic trait products require a modular and sequential multigene stacking platform that is supported by precise genome engineering technology. Designed nucleases have emerged as potent tools for creating targeted DNA double strand breaks (DSBs). Exogenously supplied donor DNA can repair the targeted DSB by a process known as gene targeting (GT), resulting in a desired modification of the target genome. The potential of GT technology has not been fully realized for trait deployment in agriculture, mainly because of inefficient transformation and plant regeneration systems in a majority of crop plants and genotypes. This challenge of transgene stacking in plants could be overcome by Intra-Genomic Homologous Recombination (IGHR) that converts independently segregating unlinked donor and target transgenic loci into a genetically linked molecular stack. The method requires stable integration of the donor DNA into the plant genome followed by intra-genomic mobilization. IGHR complements conventional breeding with genetic transformation and designed nucleases to provide a flexible transgene stacking and trait deployment platform.

A modular gene targeting system for sequential transgene stacking in plants.

A modular, selection-based method was developed for site-specific integration of transgenes into a genomic locus to create multigene stacks. High-frequency gene targeting was obtained using zinc finger nuclease (ZFN)-mediated double-strand break (DSB) formation at a pre-defined target genomic location using a unique intron directly downstream of a promoter driving a selectable marker gene to facilitate homology between target and donor sequences. In this system, only insertion into the target locus leads to a functional selectable marker, and regeneration from random insertions of the promoterless donor construct are reduced on selection media. A new stack of transgenes can potentially be loaded with each successive cycle of gene targeting by exchanging the selectable marker gene using the intron homology. This system was tested in maize using the pat selectable marker gene, whereby up to 30% of the plants regenerated on Bialaphos-containing medium were observed to have the donor construct integrated into the target locus. Unlike previous gene targeting methods that utilize defective or partial genes for selecting targeted events, the present method exchanges fully functional genes with every cycle of targeting, thereby allowing the recycling of selectable marker genes, hypothetically for multiple generations of gene targeting.