Cytokinin vectors mediate marker-free and backbone-free plant transformation

Richael, Craig; Kalyaeva, M. A.; Chretien, Robert L.; Hua, Yan; Adimulam, Sathya; Stivison, Artesia; Weeks, J. Troy; Rommens, Caius M.

doi:10.1007/s11248-008-9175-6

Cited by 32 publications

(20 citation statements)

References 42 publications

(38 reference statements)

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“…pSIM401, which only contains the selectable marker gene nptII , was used as empty vector control. The plants were transformed using a standard protocol (Richael et al. , 2008) and rooted on kanamycin‐containing medium.…”

Section: Resultsmentioning

confidence: 99%

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Tuber‐specific silencing of asparagine synthetase‐1 reduces the acrylamide‐forming potential of potatoes grown in the field without affecting tuber shape and yield

Chawla

Shakya

Rommens³

2012

Plant Biotechnology Journal

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SummarySimultaneous silencing of asparagine synthetase (Ast)-1 and -2 limits asparagine (ASN) formation and, consequently, reduces the acrylamide-forming potential of tubers. The phenotype of silenced lines appears normal in the greenhouse, but field-grown tubers are small and cracked. Assessing the effects of silencing StAst1 and StAst2 individually, we found that yield drag was mainly linked to down-regulation of StAst2. Interestingly, tubers from untransformed scions grafted onto intragenic StAst1 ⁄ 2-silenced rootstock contained almost the same low ASN levels as those in the original silenced lines, indicating that ASN is mainly formed in tubers rather than being transported from leaves. This conclusion was further supported by the finding that overexpression of StAst2 caused ASN to accumulate in leaves but not tubers. Thus, ASN does not appear to be the main form of organic nitrogen transported from leaves to tubers. Because reduced ASN levels coincided with increased levels of glutamine, it appears likely that this alternative amide amino acid is mobilized to tubers, where it is converted into ASN by StAst1. Indeed, tuber-specific silencing of StAst1, but not of StAst2, was sufficient to substantially lower ASN formation in tubers. Extensive field studies demonstrated that the reduced acrylamide-forming potential achieved by tuber-specific StAst1 silencing did not affect the yield or quality of field-harvested tubers.

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Section: Resultsmentioning

confidence: 99%

“…Stock plants were maintained in magenta boxes containing 40 mL half‐strength M516 medium (PhytoTechnology, Shawnee Mission, KS) with 3% sucrose and 2 g/L gelrite. Plants were transformed using Agrobacterium as described previously (Richael et al. , 2008).…”

Section: Methodsmentioning

confidence: 99%

Tuber‐specific silencing of asparagine synthetase‐1 reduces the acrylamide‐forming potential of potatoes grown in the field without affecting tuber shape and yield

Chawla

Shakya

Rommens³

2012

Plant Biotechnology Journal

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“…We chose the potato Ubi7 gene as our target site because it is constitutively expressed in potato plants and has a promoter that is commonly used for constitutive gene expression in potato transgenic research and applications (Richael et al, 2008). Ubi7 transcripts have an intron in the 5′ UTR.…”

Section: Resultsmentioning

confidence: 99%

“…The stable integration of the ipt expression cassette results in the overproduction of cytokinin in cells and shoots. These shoots are stunted and fail to develop roots (Richael et al, 2008), which allows for stably integrated TALEN sequences to be eliminated based on phenotype. This reduces the number of plants with stably integrated effector expression cassettes into the plant genome for further analysis.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2016

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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 transcription activator-like effector nuclease (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.

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“…Plant transformations were carried out as described previously [49]. A total of 25 RNAi lines named from 1632-1 to 1632-25 were developed for potato cultivar Ranger Russet.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2014

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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 (VInv) 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.

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Cytokinin vectors mediate marker-free and backbone-free plant transformation

Cited by 32 publications

References 42 publications

Tuber‐specific silencing of asparagine synthetase‐1 reduces the acrylamide‐forming potential of potatoes grown in the field without affecting tuber shape and yield

Tuber‐specific silencing of asparagine synthetase‐1 reduces the acrylamide‐forming potential of potatoes grown in the field without affecting tuber shape and yield

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

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

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