Selectable marker independent transformation of recalcitrant maize inbred B73 and sorghum P898012 mediated by morphogenic regulators \u3ci\u3eBABY BOOM\u3c/i\u3e and \u3ci\u3eWUSCHEL2\u3c/i\u3e

The use of morphogenic regulators to overcome barriers in plant transformation is a revolutionary breakthrough for basic plant science and crop applications. Current standard plant transformation systems are bottlenecks for genetic, genomic, and crop improvement studies. We investigated the differential use of co-expression of maize transcription factors BABY BOOM and WUSCHEL2 coupled with a desiccation inducible CRE/lox excision system to enable regeneration of stable transgenic recalcitrant maize inbred B73 and sorghum P898012 without a chemical selectable marker. The PHP78891 expression cassette contains CRE driven by the drought inducible maize RAB17M promoter with lox P sites which bracket the CRE, WUS, and BBM genes. A constitutive maize UBIM promoter directs a ZsGreen GFP expression cassette as a reporter outside of the excision sites and provides transient, transgenic, and developmental analysis. This was coupled with evidence for molecular integration and analysis of stable integration and desiccation inducible CRE-mediated excision. Agrobacterium-mediated transgenic introduction of this vector showed transient expression of GFP and induced somatic embryogenesis in maize B73 and sorghum P898012 explants. Subjection to desiccation stress in tissue culture enabled the excision of CRE, WUS, and BBM, leaving the UBIM::GFP cassette and allowing subsequent plant regeneration and GFP expression analysis. Stable GFP expression was observed in the early and late somatic embryos, young shoots, vegetative plant organs, and pollen. Transgene integration and expression of GFP positive T0 plants were also analyzed using PCR and Southern blots. Progeny segregation analysis of primary events confirmed correlation between functional GFP expression and presence of the GFP transgene in T1 plants generated from self pollinations, indicating good transgene inheritance. This study confirms and extends the use of morphogenic regulators to overcome transformation barriers

Advancing Crop Transformation in the Era of Genome Editing

Plant transformation has enabled fundamental insights into plant biology and revolutionized commercial agriculture. Unfortunately, for most crops, transformation and regeneration remain arduous even after more than 30 years of technological advances. Genome editing provides novel opportunities to enhance crop productivity but relies on genetic transformation and plant regeneration, which are bottlenecks in the process. Here, we review the state of plant transformation and point to innovations needed to enable genome editing in crops. Plant tissue culture methods need optimization and simplification for efficiency and minimization of time in culture. Currently, specialized facilities exist for crop transformation. Single-cell and robotic techniques should be developed for high-throughput genomic screens. Plant genes involved in developmental reprogramming, wound response, and/or homologous recombination should be used to boost the recovery of transformed plants. Engineering universal Agrobacterium tumefaciens strains and recruiting other microbes, such as Ensifer or Rhizobium, could facilitate delivery of DNA and proteins into plant cells. Synthetic biology should be employed for de novo design of transformation systems. Genome editing is a potential game-changer in crop genetics when plant transformation systems are optimized

Silencing of Soybean Raffinose Synthase Gene Reduced Raffinose Family Oligosaccharides and Increased True Metabolizable Energy of Poultry Feed

Soybean [Glycine max (L.) Merr.] is the number one oil and protein crop in the United States, but the seed contains several anti-nutritional factors that are toxic to both humans and livestock. RNA interference technology has become an increasingly popular technique in gene silencing because it allows for both temporal and spatial targeting of specific genes. The objective of this research is to use RNA-mediated gene silencing to down-regulate the soybean gene raffinose synthase 2 (RS2), to reduce total raffinose content in mature seed. Raffinose is a trisaccharide that is indigestible to humans and monogastric animals, and as monogastric animals are the largest consumers of soy products, reducing raffinose would improve the nutritional quality of soybean. An RNAi construct targeting RS2 was designed, cloned, and transformed to the soybean genome via Agrobacterium-mediated transformation. Resulting plants were analyzed for the presence and number of copies of the transgene by PCR and Southern blot. The efficiency of mRNA silencing was confirmed by real-time quantitative PCR. Total raffinose content was determined by HPLC analysis. Transgenic plant lines were recovered that exhibited dramatically reduced levels of raffinose in mature seed, and these lines were further analyzed for other phenotypes such as development and yield. Additionally, a precision-fed rooster assay was conducted to measure the true metabolizable energy (TME) in full-fat soybean meal made from the wild-type or transgenic low-raffinose soybean lines. Transgenic low-raffinose soy had a measured TME of 2,703 kcal/kg, an increase as compared with 2,411 kcal/kg for wild-type. As low digestible energy is a major limiting factor in the percent of soybean meal that can be used in poultry diets, these results may substantiate the use of higher concentrations of low-raffinose, full-fat soy in formulated livestock diets

Analysis of the siRNA-Mediated Gene Silencing Process Targeting Three Homologous Genes Controlling Soybean Seed Oil Quality.

In the past decade, RNA silencing has gained significant attention because of its success in genomic scale research and also in the genetic improvement of crop plants. However, little is known about the molecular basis of siRNA processing in association with its target transcript. To reveal this process for improving hpRNA-mediated gene silencing in crop plants, the soybean GmFAD3 gene family was chosen as a test model. We analyzed RNAi mutant soybean lines in which three members of the GmFAD3 gene family were silenced. The silencing levels of FAD3A, FAD3B and FAD3C were correlated with the degrees of sequence homology between the inverted repeat of hpRNA and the GmFAD3 transcripts in the RNAi lines. Strikingly, transgenes in two of the three RNAi lines were heavily methylated, leading to a dramatic reduction of hpRNA-derived siRNAs. Small RNAs corresponding to the loop portion of the hairpin transcript were detected while much lower levels of siRNAs were found outside of the target region. siRNAs generated from the 318-bp inverted repeat were found to be diced much more frequently at stem sequences close to the loop and associated with the inferred cleavage sites on the target transcripts, manifesting "hot spots". The top candidate hpRNA-derived siRNA share certain sequence features with mature miRNA. This is the first comprehensive and detailed study revealing the siRNA-mediated gene silencing mechanism in crop plants using gene family GmFAD3 as a test model

Putative functional siRNAs.

<p>^a Sequence is ranked from CPM high to low in S-24-4D.</p><p>^b CPM is average of three biological replications.</p><p>Putative functional siRNAs.</p

5’RACE on Gm<i>FAD3A</i> and Gm<i>FAD3C</i> mRNAs in T₅ RNAi lines.

<p>Arrows indicate the inferred cleavage sites and numbers above represent the fractions of cloned 5’ RACE PCR products terminating at this position. Degradation sites detected with high frequency are highlighted in red, and those present across the three RNAi lines are highlighted with asterisks. (A) Summary of the 5’ RACE analysis performed on the 318-bp region of Gm<i>FAD3A</i> mRNA. (B) and (C) Summary of the 5’ RACE analysis performed on the corresponding region of Gm<i>FAD3C</i> mRNA.</p

Comparison of 318-bp IR-derived siRNAs in the three RNAi lines.

<p>(A) Small RNAs matching the 318-bp IR were plotted versus the average of their normalized abundance from three replications. Plus Y-axis labels represent siRNAs from the sense strand of 318-bp region, while minus Y-axis indicate siRNAs found on the opposite strand. For visual clarity, the Y-axis of each diagram is adjusted based on the corresponding small RNA abundance. (B) and (C) Venn diagram represents common and specific reads from total and top 1000 abundant small RNAs in S-24-4D, S-24-13 and S-24-15, respectively.</p

DNA methylation analysis of T₅ homozygous RNAi lines.

<p><b>(A)</b> Schematic presentation of the T-DNA region of the plant transformation vector, pMUFAD. The expression cassette for the RNAi of <i>GmFAD3</i> is highlighted in grey. Note: LB and RB, T-DNA left and right borders, respectively; Tvsp, soybean vegetative storage protein gene terminator; <i>bar</i>, bialaphos resistance gene; TEV, tobacco etch virus translational enhancer; CaMV35S, cauliflower mosaic virus 35S promoter; OCS 3’, octopine synthase gene terminator; IR-R and IR-F, the 318-bp inverted repeats of <i>GmFAD3</i> target sequence in reverse and forward directions, respectively; Rice Waxy-a Intron, rice Waxy-a gene intron; GlyP, soybean glycinin gene promoter. Lines beneath the schematic represent region I, II, III and IV examined by bisulfite sequencing. <b>(B)</b> Schematic of the <i>Glycinin</i> gene (GenBank: AB113349.1). The darkened rectangle represents exon and horizontal line represents intron. The black arrow indicates transcription starting site. Lines beneath the schematic represent region V and VI examined by bisulfite sequencing, with numbers indicating the corresponding position. <b>(C)</b> Methylation status of a 338 bp region I (294bp Glycinin promoter, 32bp vector backbone, 12bp forward inverted-repeat of the <i>FAD3</i> hairpin), a 276bp region II, a 281bp region III and a 352bp region IV within the plant transformation vector pMUFAD. (D) Methylation status of a 297 bp region V (-295 bp to 2bp) and a 255 bp region VI (1105 bp to 1359bp) within soybean <i>Glycinin</i> gene (GenBank: AB113349.1). Bar heights represent the percentage of methylation at each CGN, CHG and CHH (where N = A, T, G or C; H = A, T, or C) cytosines of 10 clones analyzed by bisulfite sequencing. Two biological replications were performed and similar results were obtained.</p