Maximizing the expression of transgenic traits into elite alfalfa germplasm using a supertransgene configuration in heterozygous conditions

Jozefkowicz, Cintia; Frare, Romina; Fox, Ana Romina; Odorizzi, Ariel; Arolfo, Valeria; Pagano, Elba; Basigalup, Daniel Horacio; Ayub, Nicolás Daniel; Soto, Gabriela

doi:10.1007/s00122-018-3062-1

Cited by 11 publications

(7 citation statements)

References 31 publications

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“…(6) Alfalfa is generally self-incompatible, allogamous, and several insects can pollinate its flowers (Monteros et al, 2014). A little progress has been achieved in the incorporation of desired transgenic traits into commercial alfalfa although the ecological and economic importance of the allogamous and autotetraploid alfalfa crop (Jozefkowicz et al, 2018).…”

Section: Constraints Of Production and Handling Of Alfalfamentioning

confidence: 99%

Sustainable Biorefinery and Production of Alfalfa (Medicago sativa L.)

et al. 2020

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Egyptian Journal of Botany http://ejbo.journals.ekb.eg/ Review 45 A LFALFA is considered as "the Queen of the Forages" due to its high protein content and nutritional value. The production of alfalfa under different stressful environments is a great challenge, representing a serious threat to global food security. To improve the production of alfalfa under stressful conditions, there is a crucial need to understand the response of alfalfa plants to stresses, the mechanisms of tolerance and the management options. Alfalfa is a promising biorefinery crop from which leaf protein concentrates as well as bio-organic fertilizers can be produced. The potential of leaf-derived protein concentrate for human and animal nutrition is huge, yet unexploited. The bio-organic fertilizers derived from alfalfa plants represent a sustainable way to supply the plant with some nutrients. This review article highlights the sustainable use of alfalfa in biorefinery and bio-organic fertilizer production. Moreover, scattered knowledge about its use for production is collected supporting its suitability for improving soil fertility. Last, we discuss the suitability of alfalfa to produce stable yields in the light of changing climate and stressful conditions like salinity.

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Section: Constraints Of Production and Handling Of Alfalfamentioning

confidence: 99%

Sustainable Biorefinery and Production of Alfalfa (Medicago sativa L.)

et al. 2020

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“…Since cultivated alfalfa is a tetraploid, allogamous, perennial species, species-specific factors influence the survival and high production of an alfalfa stand (e.g., plant regrowth), which depends on complex allelic variants. Besides, although alfalfa exhibits high levels of self-incompatibility, the homozygosis in an individual locus can be rapidly and inexpensively generated via alfalfa selfing by using non-emasculated hand crosses under greenhouse conditions (Jozefkowicz et al, 2018). Consequently, full allelic knock-out of an individual alfalfa gene could be obtained by autofecundation of edited plants, independently of the efficiency of the CRISPR/Cas9 system to simultaneously target multiple alleles in this crop.…”

Section: Resultsmentioning

confidence: 99%

“…For the insertion and mutation of the GUS gene in the alfalfa genome, two transgenic alfalfa events, called here 3-1 and 5-1, which show high and constitutive expression of the GUS reporter, were generated by transforming the regenerative alfalfa clone C2-3 with the binary vector pBI121 (Accession Number: AF485783.1) as described previously (Garcia et al, 2014;Jozefkowicz et al, 2018). To determine the number of T-DNA insertions in 3-1 and 5-1, these 35S-GUS-transformed alfalfa events were individually crossed manually with CW1010 wild-type alfalfa by using transgenic parental plants as pollen donors.…”

Section: Methodsmentioning

confidence: 99%

“…Consequently, there is a continuing and increasing interest in the development of alternative methods for the editing of alfalfa genome, which partially depends on the regenerative genotype used during alfalfa transformation (Gao et al, 2018;Chen et al, 2020;Wolabu et al, 2020). Previously, we developed a highly effective procedure for the transformation of the public alfalfa clone C23 via Agrobacterium tumefaciens (Garcia et al, 2014;Jozefkowicz et al, 2016Jozefkowicz et al, , 2018Stritzler et al, 2018;Pascuan et al, 2020). In this brief research report, we used this framework for the production of CRISPR/Cas9 gene-edited alfalfa from clone C23.…”

Section: Introductionmentioning

confidence: 99%

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Efficient CRISPR/Cas9 Genome Editing in Alfalfa Using a Public Germplasm

et al. 2021

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Because its ability to acquire large amounts of nitrogen by symbiosis, tetraploid alfalfa is the main source of vegetable proteins in meat and milk production systems in temperate regions. Alfalfa cultivation also adds fixed nitrogen to the soil, improving the production of non-legumes in crop rotation and reducing the use of nitrogen fertilizers derived from fossil fuel. Despite its economic and ecological relevance, alfalfa genetics remains poorly understood, limiting the development of public elite germplasm. In this brief article, we reported the high-efficiency of alfalfa mutagenesis by using the public clone C23 and the CRISPR/Cas9 system. Around half of the GUS overexpressing plants (35S-GUS under C23 genomic background) transformed with an editing plasmid containing two sgRNAs against the GUS gene and the Cas9 nuclease exhibited absence of GUS activity. Nucleotide analysis showed that the inactivation of GUS in CRISPR/Cas9-editing events were produced via different modifications in the GUS gene, including frameshift and non-sense mutations. Using the CRISPR/Cas9 system and two sgRNAs, we have also edited the alfalfa gene NOD26, generating plants with different doses of alleles at this locus, including complete gene knockout at high efficiency (11%). Finally, we discuss the potential applications of genome-editing technologies to polyploid research and to alfalfa improvement public programs.

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“…S5), cloned in the pGEM-easy vector, sequenced and subcloned in the binary vector pBI121 (García, et al 2014). We produced stable transformants that carry the pMtNOD26a-GUS and pMtNOD26b-GUS fusions by using wild-type regenerative clone C2-3 as previously (Jozefkowicz, et al 2018;Pascuan, et al 2020). Kanamycin-tolerant plants derived from different calluses were considered independent transgenic events.…”

Section: Production and Characterization Of Transcriptional Fusion Linesmentioning

confidence: 99%

The nodule-specific multifunctional channel NOD26 of Medicago truncatula is crucial for nitrogen-fixing symbiosis

Frare¹,

Gómez²,

López-Fernández³

et al. 2021

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The multifunctional channel NOD26, identified and extensively studied (both biochemically and biophysically) in soybean, is a major protein component of the symbiosome membrane. The water and ammonia transport activities of NOD26 are thought to be important for nodule development, osmotic balance, and ammonia efflux from the symbiosome. However, the widely accepted relevance of NOD26 in nitrogen-fixing symbiosis has never been explored in planta. Recently, we have reported the emergence of NOD26 in the nitrogen-fixing clade of angiosperms via tandem duplication. Here, we characterized the two copies of NOD26 from Medicago truncatula (Medtr8g087710 and Medtr8g087720) in their transport abilities, and at gene expression and genetic levels. Similar to their homologous soybean gene, MtNOD26 genes encode water and ammonia transport activities in heterologous expression systems. By using multiple transcriptional studies (RT-qPCR, transcriptional fusion and RNA-Seq analyses), we found that the expression of MtNOD26 copies is restricted to the nodule and gradually increases from the bacteria-free meristematic region to the nitrogen-fixation zone. Under nitrogen-limiting soil conditions, the homozygous insertional mutant lines of these two MtNOD26 genes had the same aberrant nodulation phenotype and chlorosis. Similar to uninoculated wild-type plants, inoculated mutants were unable to grow in minimal medium without a nitrogen source. Collectively, our findings suggest functional equivalence between MtNOD26 copies and underline a crucial role of NOD26 in symbiotic nitrogen fixation.

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Maximizing the expression of transgenic traits into elite alfalfa germplasm using a supertransgene configuration in heterozygous conditions

Cited by 11 publications

References 31 publications

Sustainable Biorefinery and Production of Alfalfa (Medicago sativa L.)

Sustainable Biorefinery and Production of Alfalfa (Medicago sativa L.)

Efficient CRISPR/Cas9 Genome Editing in Alfalfa Using a Public Germplasm

The nodule-specific multifunctional channel NOD26 of Medicago truncatula is crucial for nitrogen-fixing symbiosis

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