Sonication-assisted Agrobacterium-mediated transformation of the ACC gene to interfere the production of ethylene in spring Dendrobium cv. ‘Sanya’

Sonication-assisted Agrobacterium-mediated transformation of the ACC gene to interfere the... In the present study, a protocol was developed to obtain stable lines of the spring Dendrobium cv. ‘Sanya’ via Sonication-Assisted Agrobacterium-mediated Transformation (SAAT) of protocorm-like bodies (PLBs). Agrobacterium tumefaciens strain LBA4404 was used with the binary vector YF9078 containing the inverted repeat (IR) of ACC gene (IRACC) for the inhibition of ethylene production, the neomycin phosphotransferase II gene (NPTII) gene as a selectable marker for kanamycin resistance, and an intron-containing β-glucuronidase gene (gus-int) as a reporter gene. PLBs were treated for 10 min with ultrasound (40 kHz) and subsequently inoculated with Agrobacterium suspension for 60 min. The addition of 100 μM acetosyringone (AS) during the preculturing, inoculation and co-cultivation resulted in the highest efficiency of gene transformation (1.04%). Following co-cultivation, PLBs were cultured on selective medium containing 200 mg/l kanamycin and 250 mg/l cefotaxime. Proliferating PLBs were repeatedly selected for kanamycin resistance, and stable transformed lines were generated. Incorporation and expression of transgenes were confirmed by GUS histochemical assay, PCR analysis, Southern blot analysis, and real-time reverse transcription-polymerase chain reaction (RT-PCR). A total of five Southern blot positive transgenic lines were obtained. The ability for ethylene suppression by small potted transgenic plants was measured. The results showed that it was significantly higher in transgenic lines than in untransformed control; different strains exhibited marked variation; and this was related to their cDNA expression. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Russian Journal of Plant Physiology Springer Journals

Sonication-assisted Agrobacterium-mediated transformation of the ACC gene to interfere the production of ethylene in spring Dendrobium cv. ‘Sanya’

Loading next page...
 
/lp/springer_journal/sonication-assisted-agrobacterium-mediated-transformation-of-the-acc-bMi5JFCohG
Publisher
Springer Journals
Copyright
Copyright © 2012 by Pleiades Publishing, Ltd.
Subject
Life Sciences; Plant Sciences; Plant Physiology
ISSN
1021-4437
eISSN
1608-3407
D.O.I.
10.1134/S1021443712010190
Publisher site
See Article on Publisher Site

Abstract

In the present study, a protocol was developed to obtain stable lines of the spring Dendrobium cv. ‘Sanya’ via Sonication-Assisted Agrobacterium-mediated Transformation (SAAT) of protocorm-like bodies (PLBs). Agrobacterium tumefaciens strain LBA4404 was used with the binary vector YF9078 containing the inverted repeat (IR) of ACC gene (IRACC) for the inhibition of ethylene production, the neomycin phosphotransferase II gene (NPTII) gene as a selectable marker for kanamycin resistance, and an intron-containing β-glucuronidase gene (gus-int) as a reporter gene. PLBs were treated for 10 min with ultrasound (40 kHz) and subsequently inoculated with Agrobacterium suspension for 60 min. The addition of 100 μM acetosyringone (AS) during the preculturing, inoculation and co-cultivation resulted in the highest efficiency of gene transformation (1.04%). Following co-cultivation, PLBs were cultured on selective medium containing 200 mg/l kanamycin and 250 mg/l cefotaxime. Proliferating PLBs were repeatedly selected for kanamycin resistance, and stable transformed lines were generated. Incorporation and expression of transgenes were confirmed by GUS histochemical assay, PCR analysis, Southern blot analysis, and real-time reverse transcription-polymerase chain reaction (RT-PCR). A total of five Southern blot positive transgenic lines were obtained. The ability for ethylene suppression by small potted transgenic plants was measured. The results showed that it was significantly higher in transgenic lines than in untransformed control; different strains exhibited marked variation; and this was related to their cDNA expression.

Journal

Russian Journal of Plant PhysiologySpringer Journals

Published: Feb 26, 2012

References

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off