Improvement of Agrobacterium-mediated transformation of cucumber (Cucumis sativus L.) by combination of vacuum infiltration and co-cultivation on filter paper wicks

Nanasato, Yoshihiko; Konagaya, Ken-ichi; Okuzaki, Ayako; Tsuda, Masayuki; Tabei, Yutaka

doi:10.1007/s11816-012-0260-1

Cited by 52 publications

(55 citation statements)

References 31 publications

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“…Vacuum infiltration treatment was performed as previously described (Nanasato et al 2013). Wounded explants were placed in a bioreactor tube (TPP TubeSpin; 50 ml; TPP Cell Culture Plastics, St. Louis, MO) containing 20 ml of the Agrobacterium inoculum.…”

Section: Inoculation Condition and Screening Transgenic Plantsmentioning

confidence: 99%

Improving the transformation efficiency of Cucurbita species: factors and strategy for practical application

Nanasato

Okuzaki

Tabei

2013

Plant Biotechnology

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Cucurbita species are refractory to transformation. Hence, the only 2 reports published regarding the transformation of Cucurbita species until our successful transformation of C. moschata (cv. Heiankogiku) in 2011. The efficiency was 2.7±1.3% using wounded explants vortexed with whisker suspension. To improve transformation efficiency, transformation experiments were carried out with various ages of cotyledonary explants. The highest efficiency was obtained with 1-day-old (3.2±0.9%) and 2-day-old (3.3±0.8%) explants after germination. Histochemical analysis of GUS activity revealed that wounding allowed Agrobacterium access to the deeper layer of explants. These results suggested that cells having a high ability of shoot organogenesis exist not on the surface but in the deeper layers of explants. We applied vacuum infiltration to wounded explants to enhance Agrobacterium access to the deeper layers, which resulted in improvement of transformation efficiency by 3-fold (9.2±2.9%). To verify the efficacy of our transformation procedure for other Cucurbita species, we attempted to obtain transformants with 3 Cucurbita species: C. maxima (cv. Ebisu), C. pepo (cv. Black Tosca), and C. ficifolia (cv. Kurotanenankin). The average regeneration efficiencies were 54.2±7.2%, 62.5±19.1%, and 72.2±13.4% under our regeneration system. Although the efficiency for production of tansgenic plants was very low (ca. 0.2-0.3%), a transgenic line was obtained from C. maxima and C. pepo, respectively. We discuss recent advances that may help in the development of beneficial applications for the transformation of Cucurbita species.

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Section: Inoculation Condition and Screening Transgenic Plantsmentioning

confidence: 99%

Improving the transformation efficiency of Cucurbita species: factors and strategy for practical application

Nanasato

Okuzaki

Tabei

2013

Plant Biotechnology

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“…In this protocol, a standard final concentration of 100 µM acetosyringone was added just prior to inoculation. After infection, callus were blot dry and kept on filter paper wicks rather than solid media, based on earlier reports that filter paper wicks increase Agrobacterium infection efficiency [37] [38]. After co-cultivation, callus is kept on resting media to avoid over-growth for 7 -10 days in the callus induction media without hygromycin selection so that callus regains their proliferating capacity.…”

Section: Production Of Osa-mir820 Over-expressing Ricementioning

confidence: 99%

Protocol for Artificial MicroRNA Mediated Over-Expression of miR820 in Indica Rice

Sharma¹,

Panchal²,

Sanan‐Mishra³

2015

AJPS

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In the present study, we illustrate the strategy and protocol required to generate rice transgenics over-expressing the 21-nt form of Osa-miR820. The miR exists in two size variants of 21-nt and 24-nt so the natural precursor cannot be employed for the purpose of miR over-expression as the cellular machinery can process both size variants thereby masking the role of PTGS regulation. Hence, we adopted the artificial miR technology to specifically over-express the 21-nt species in the transgenics. During the course of experiments it was observed that the amiR constructs probably interfered with the regeneration of the transformed callus, necessitating protocol modifications. The results indicate the successful over-expression of the 21-nt miR species. These plants can serve as a useful source for the functional dissection of the role played by the 21-nt Osa-miR820 species. They will also be valuable in highlighting the importance for the existence of a dual mode of miR mediated target regulation.

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“…The Agrobacterium cells were cultured overnight at 28°C in 50 ml tubes containing liquid Luria broth (pH 5.2) supplemented with 25 mg l −1 gentamicin, 25 mg l 2,4-d, 10 mM MES, 3% sucrose, pH 5.2) supplemented with 200 µM acetosyringone. The suspended cells were kept for 2 h at room temperature to ensure efficient induction of vir genes (Nanasato et al 2011(Nanasato et al , 2012.…”

Section: Bacterial Strainsmentioning

confidence: 99%

Application of the acetolactate synthase gene as a cisgenic selectable marker for Agrobacterium-mediated transformation in Chinese cabbage (Brassica rapa ssp. pekinensis)

Konagaya

Tsuda

Okuzaki

et al. 2013

Plant Biotechnology

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Heading Chinese cabbage (Brassica rapa ssp. pekinensis) has been recalcitrant to regeneration and transformation. In particular, there are few reports concerning Japanese cultivars. We evaluated the factors that inhibit Agrobacterium-mediated transformation in heading Chinese cabbage. Then, we investigated the effects of selectable markers using heading Chinese cabbage cv. Chihiri 70 and compared the effects with those in broccoli cv. Ryokurei (B. oleracea var. italica) as a control for an easily transformable Brassica species. To utilize a selectable marker derived from a host plant cisgene, we cloned a genomic DNA fragment containing regulatory and coding sequences of the acetolactate synthase (ALS) gene from Chinese cabbage and mutagenized it to a herbicide resistant form. After transformation of Chinese cabbage and broccoli with this construct, transgenic plants were efficiently selected with the herbicide bispyribac sodium salt and screened by DNA gel blot analysis. The average transformation frequency of Chinese cabbage was 1.2±0.2%, which was similar to those in reports using antibiotic selectable markers and was lower than for broccoli (13.9±2.0%). Furthermore, the escape rate was restricted at a low level (about 35-50% lower than hygromycin selection), which is an advantage on practical transformation. We confirmed transgene inheritance and herbicide resistance of potted plants in the T 1 generation. This report is the first to describe a selection system for the transformation of a Brassica crop that uses a herbicide-tolerant selectable marker derived from a cisgene.

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Improvement of Agrobacterium-mediated transformation of cucumber (Cucumis sativus L.) by combination of vacuum infiltration and co-cultivation on filter paper wicks

Cited by 52 publications

References 31 publications

Improving the transformation efficiency of Cucurbita species: factors and strategy for practical application

Improving the transformation efficiency of Cucurbita species: factors and strategy for practical application

Protocol for Artificial MicroRNA Mediated Over-Expression of miR820 in Indica Rice

Application of the acetolactate synthase gene as a cisgenic selectable marker for Agrobacterium-mediated transformation in Chinese cabbage (Brassica rapa ssp. pekinensis)

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