Highly Efficient <i>Agrobacterium</i>‐Mediated Transformation of Potato (<i>Solanum tuberosum</i>) and Production of Transgenic Microtubers

Craze, Melanie; Bates, Ruth; Bowden, Sarah; Wallington, Emma J.

doi:10.1002/cppb.20065

Cited by 13 publications

(10 citation statements)

References 12 publications

(11 reference statements)

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“…The implementation of a regeneration procedure for ‘Yagana-INIA’ was based on previous descriptions in ‘Désirée’ [ 23 ]. After adjustments, we observed that the ‘Yagana-INIA’ explants responded to the respective culture media with a progressive delay as development stages advanced.…”

Section: Resultsmentioning

confidence: 99%

“…Positive clones were purified using the Zymo Miniprep Kit (Zymo Research) and sequenced (Macrogen Inc.) to verify a correct assembly, also using the gmv-CI-Fw/gmv-CI-Rv primer pair. pGEF-X clones were electroporated into electrocompetent Agrobacterium tumefaciens strain EHA105 as described above and used to transform leaf explants of ‘Yagana-INIA’, according to the previously described procedure [ 23 ].…”

Section: Methodsmentioning

confidence: 99%

“…4.3.3. Explants' Infection, Co-Culture, and Callus Induction Infections were as described in Craze et al [23] with modifications. Pre-cultured explants were incubated in the activated Agrobacterium solution (30 mL PLM plus AS 200 µM) for 30 min with gentle agitation (50-70 rpm).…”

Section: Explants Pre-culturementioning

confidence: 99%

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A Traceable DNA-Replicon Derived Vector to Speed Up Gene Editing in Potato: Interrupting Genes Related to Undesirable Postharvest Tuber Traits as an Example

Acha

Vergara

Muñoz

et al. 2021

Plants

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In potato (Solanum tuberosum L.), protoplast techniques are limited to a few genotypes; thus, the use of regular regeneration procedures of multicellular explants causes us to face complexities associated to CRISPR/Cas9 gene editing efficiency and final identification of individuals. Geminivirus-based replicons contained in T-DNAs could provide an improvement to these procedures considering their cargo capability. We built a Bean yellow dwarf virus-derived replicon vector, pGEF-U, that expresses all the editing reagents under a multi-guide RNA condition, and the Green Fluorescent Protein (GFP) marker gene. Agrobacterium-mediated gene transfer experiments were carried out on ‘Yagana-INIA’, a relevant local variety with no previous regeneration protocol. Assays showed that pGEF-U had GFP transient expression for up to 10 days post-infiltration when leaf explants were used. A dedicated potato genome analysis tool allowed for the design of guide RNA pairs to induce double cuts of genes associated to enzymatic browning (StPPO1 and 2) and to cold-induced sweetening (StvacINV1 and StBAM1). Monitoring GFP at 7 days post-infiltration, explants led to vector validation as well as to selection for regeneration (34.3% of starting explants). Plant sets were evaluated for the targeted deletion, showing individuals edited for StPPO1 and StBAM1 genes (1 and 4 lines, respectively), although with a transgenic condition. While no targeted deletion was seen in StvacINV1 and StPPO2 plant sets, stable GFP-expressing calli were chosen for analysis; we observed different repair alternatives, ranging from the expected loss of large gene fragments to those showing punctual insertions/deletions at both cut sites or incomplete repairs along the target region. Results validate pGEF-U for gene editing coupled to regular regeneration protocols, and both targeted deletion and single site editings encourage further characterization of the set of plants already generated.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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A Traceable DNA-Replicon Derived Vector to Speed Up Gene Editing in Potato: Interrupting Genes Related to Undesirable Postharvest Tuber Traits as an Example

Acha

Vergara

Muñoz

et al. 2021

Plants

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“…and the GA 3 also had different concentration in different species. The results of others research also indicated that the hormone ratio required for different genotypes of potato to differentiate into shoots was different, and the differentiation efficiency was similarly different [21][22][23][24][25].…”

Section: Selection Of Shoots Induction Medium For Callusmentioning

confidence: 99%

Construction of Potato DM1-3-516-R44 (DM) Transgenic System Based on Agrobacterium Transformation

Zhang

Wang

Yang

et al. 2020

Preprint

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Background: The sequencing potato DM1-3-516-R44 played an irreplaceable role in the study of gene function. So far, no one research the transformation system about DM. Therefore, our experiment was studied from three aspects: plant regeneration system, optimization of agrobacterium infection conditions and the effect of hygromycin on DM. Results: A relatively suitable method for genetic transformation of DM was obtained: 1) The stem callus induction medium was MS + IAA 0.5 mg/L + 6-BA 2.0 mg/L, the leaf callus induction medium was MS + NAA 1.0mg/L + 6-BA 0.5mg/L and the shoot differentiation medium was MS + 6-BA 3.0 mg/L + ZT 0.5 mg/L. 2) The specific transformation condition was the agrobacterium concentration kept the OD600 = 0.3, and co-culture time consisted 3 days in the dark. The hygromycin concentration chose 8 mg/L to screen the transgenic plants. 3) Using hygromycin to screen about 100 transgenic shoots, 75 shoots were obtained and 53 strains were identified had target stripe by PCR technology. Conclusion: The efficiency of the transformation system we created was over 50%. It provided a good basis for the study of potato gene function.

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“…Potato is primitively domesticated and consumed in North America, Europe, former USSR nations, and now a large number of cultivations are carried out in South Africa, Asia, and South America. This makes potato the world's fourth most imperative crop in terms of production (Hong et al, 2017;Craze et al, 2018). It has been reported that worldwide potato production was 388,190,674 tonnes with the area cultivated on 19,302,642 hectares (FAOSTAT, 2017).…”

Section: Introductionmentioning

confidence: 99%

Manipulating genome of diploid potato inbred line Solanumchacoense M6 using selectable marker gene

Dangol¹,

Yel²,

Çalışkan³

et al. 2020

Turk J Agric For

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The development of transgenic potatoes is imperative to investigate various gene functions as well as to develop robust potato varieties resistant to different biotic and abiotic stresses. Directing a potato breeding program from cultivated tetraploids to selfcompatible diploid lines will be highly helpful for potato breeders. However, diploid potatoes are considered recalcitrant to regeneration. We aimed to develop a protocol for the transformation of diploid Solanum chacoense M6 potatoes using leaf, internodes, and microtubers as the explants. Agrobacterium tumefaciens strain GV2260 harboring pBIN19 expression vector containing gusA gene (interrupted by an intron to induce expression from eukaryotic cells) was used for this purpose. Different inoculation times (10, 20, 30 min) were applied in aforementioned explants for gene transfer. After cocultivation, explants were transferred to the media with various hormone concentrations [6-benzylaminopurine (BAP), 1-naphthaleneacetic acid (NAA), trans-zeatin, kinetin, and 2, 4-Dichlorophenoxyacetic acid (2,4-D)]. The calli generated were then transferred to shoot generating medium supplemented with thidiazuron (TDZ) and gibberellic acid (GA 3 ). According to histochemical GUS analysis, we found a 20 min inoculation time to be optimal for gene transfer and the medium containing 2 mg L -1 BAP and 2 mg L -1 NAA was the most suitable medium for callus induction from 20 min inoculated explants (41% callus formation for internodes and 65% for leaf explants). Abundant transcripts levels of gusA gene was found in primary transformants when subjected to RT-qPCR. GUS fluorometric assay further confirmed the primary transformants at protein level. The present study can serve as a gateway to transfer gene(s) of interest in diploid potatoes.

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Highly Efficient Agrobacterium‐Mediated Transformation of Potato (Solanum tuberosum) and Production of Transgenic Microtubers

Cited by 13 publications

References 12 publications

A Traceable DNA-Replicon Derived Vector to Speed Up Gene Editing in Potato: Interrupting Genes Related to Undesirable Postharvest Tuber Traits as an Example

A Traceable DNA-Replicon Derived Vector to Speed Up Gene Editing in Potato: Interrupting Genes Related to Undesirable Postharvest Tuber Traits as an Example

Construction of Potato DM1-3-516-R44 (DM) Transgenic System Based on Agrobacterium Transformation

Manipulating genome of diploid potato inbred line Solanumchacoense M6 using selectable marker gene

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