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Euphytica

, Volume 177, Issue 1, pp 33–43 | Cite as

Genetic transformation of lipid transfer protein encoding gene in Phalaenopsis amabilis to enhance cold resistance

  • Xuyin Qin
  • Yang Liu
  • Shanjing Mao
  • Tianbi Li
  • Hsinkan Wu
  • Chengcai Chu
  • Youping Wang
Article

Abstract

Embryogenic callus of Phalaenopsis amabilis derived from leaf tissue was cocultivated with Agrobacterium tumefaciens strain LBA4404 harboring a plant cloning vector. The vector carried the lipid transfer protein (LTP) encoding gene cloned from cold tolerant Brazilian upland rice cv. IAPAR 9. The highest transformation efficiency (12.16%) was obtained when 1–2 mm calli were infected and cocultivated with 0.4 (OD600) A. tumefaciens for 20 min. Transgene integration of kan-resistant plants was confirmed through polymerase chain reaction analysis and Southern hybridization. Four hundred seventy transgenic plants, each derived from an independent protocorm-like body, were obtained. The expression of rice cold-inducible LTP gene in transgenic P. amabilis improved its adaptive responses to cold stress. The examination of transgenic plants revealed that enhanced cold tolerance was most likely due to the increased accumulation of several compatible solutes such as total soluble sugars, proline, antioxidant superoxide dismutase, decreased accumulation of malondialdehyde, and maintained electrolytes within the membrane compared with controls.

Keywords

Phalaenopsis amabilis Agrobacterium tumefaciens-mediated transformation LTP gene Cold tolerance 

Abbreviations

ANOVA

Analysis of variance

AS

Acetosyringone

BAP

6-Benzylaminopurine

Cef

Cefotaxime

EDTA

Ethylene diaminetetraacetic acid

EL

Electrolyte leakage

Kan

Kanamycin

LB

Lauria bertani

LTP

Lipid transfer protein

MDA

Malondialdehyde

MET

Methionine

MS

Murashige and Skoog medium

NAA

Naphthalene acetic acid

NBT

Nitroblue tetrazolium

npt II

Neomycin phosphotransferase II

PCR

Polymerase chain reaction

PLB

Protocorm-like body

pUbi

Plant ubiquitin promoter

SDW

Sterile distilled water

SDS

Sodium dodecyl sulfate

SOD

Superoxide dismutase

TBA

Thiobarbituric acid

TDZ

Thidiazuron

TE

Transformation efficiency

VB6

Pyridoxine HCl

Notes

Acknowledgments

This work was funded by Chinese National Major Program of Transgenic Research (2009ZX08004-002B).

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Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Xuyin Qin
    • 1
  • Yang Liu
    • 1
  • Shanjing Mao
    • 1
  • Tianbi Li
    • 1
  • Hsinkan Wu
    • 2
  • Chengcai Chu
    • 3
  • Youping Wang
    • 1
  1. 1.College of Bioscience and BiotechnologyYangzhou UniversityYangzhouChina
  2. 2.Yangping Horticulture Co. LtdYangzhouChina
  3. 3.Institute of Genetics and Developmental BiologyChinese Academy of SciencesBeijingChina

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