Transcription Activator-Like Effector Nucleases (TALEN)-Mediated Targeted DNA Insertion in Potato Plants

Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. Specifically integrated transgenes are guaranteed to co-segregate, and expression level is more predictable, which makes downstream characterization and line selection more manageable. Because the site of DNA integration is known, the steps to deregulation of transgenic crops may be simplified. Here we describe a method that combines TALEN-mediated induction of double strand breaks (DSBs) and non-autonomous marker selection to insert a transgene into a pre-selected, transcriptionally active region in the potato genome. In our experiment, TALEN was designed to create a DSB in the genome sequence following an endogenous constitutive promoter. A cytokinin vector was utilized for TALENs expression and prevention of stable integration of the nucleases. The donor vector contained a gene of interest cassette and a promoter-less plant-derived herbicide resistant gene positioned near the T-DNA left border which was used to select desired transgenic events. Our results indicated that TALEN induced T-DNA integration occurred with high frequency and resulting events have consistent expression of the gene of interest. Interestingly, it was found that, in most lines integration took place through one sided homology directed repair despite the minimal homologous sequence at the right border. An efficient transient assay for TALEN activity verification is also described

Vacuolar Invertase Gene Silencing in Potato (<i>Solanum tuberosum</i> L.) Improves Processing Quality by Decreasing the Frequency of Sugar-End Defects

<div><p>Sugar-end defect is a tuber quality disorder and persistent problem for the French fry processing industry that causes unacceptable darkening of one end of French fries. This defect appears when environmental stress during tuber growth increases post-harvest vacuolar acid invertase activity at one end of the tuber. Reducing sugars produced by invertase form dark-colored Maillard reaction products during frying. Acrylamide is another Maillard reaction product formed from reducing sugars and acrylamide consumption has raised health concerns worldwide. Vacuolar invertase gene (<i>VInv</i>) expression was suppressed in cultivars Russet Burbank and Ranger Russet using RNA interference to determine if this approach could control sugar-end defect formation. Acid invertase activity and reducing sugar content decreased at both ends of tubers. Sugar-end defects and acrylamide in fried potato strips were strongly reduced in multiple transgenic potato lines. Thus vacuolar invertase silencing can minimize a long-standing French fry quality problem while providing consumers with attractive products that reduce health concerns related to dietary acrylamide.</p></div

Sugar-end defect frequency was reduced in French fries prepared from <i>VInv</i>-silencing lines of Ranger Russet.

<p>Sugar-end defects are apparent on nearly half of the fries from Ranger Russet (A) and empty vector (B) control tubers. No sugar-end defect fries where observed in fries from lines 1632-1 (C) and 1632-4 (D) in which the <i>VInv</i> had been silenced using RNA interference. In (A) and (B), fries with sugar-end defects are on the right and fries without sugar-end defects are on the left.</p

Expression of <i>VInv</i> in the bud end and stem end of tubers from <i>VInv</i>-silencing lines of Russet Burbank (RBKx) relative to expression of <i>VInv</i> in untransformed Russet Burbank at harvest and after one, three and five months of storage.

<p>Note: ^§Asterisks indicate overall differences of least squares means between RBKx and Russet Burbank controls at the same tuber end across sampling times of harvest and one, three and five months of storage.</p><p>*, p<0.05;</p><p>**, p<0.01;</p><p>***, p<0.001.</p><p><i>VInv</i> expression was determined using <i>actin97</i> as a reference gene and results are presented as a percentage of the level in Russet Burbank controls.</p

Acrylamide content at stem end and bud end of fried strips prepared from Russet Burbank and three <i>VInv</i>-silencing lines three months after harvest.

<p>Note: Acrylamide contents are means ± standard deviation of five individual fried strips.</p><p>Note that lines RBK1 and RBK22 have strong suppression of <i>VInv</i> and RBK46 shows little suppression of <i>VInv</i>.</p

Sucrose content in both ends of Russet Burbank potato tubers and in tubers from <i>VInv</i>-silencing lines (RBKx) of Russet Burbank at harvest and after one, three and five months of storage.

<p>Note: Data are means ± standard deviation of five independent tuber samples. Sucrose contents are shown as mg g⁻¹ fresh tuber weight.</p