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

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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.

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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

References

  • Anchordoguy TJ, Rudolph AS, Carpenter JF, Crowe JH (1987) Modes of interaction of cryoprotectants with membrane phospholipids during freezing. Cryobiology 24:324–331

    Article  CAS  PubMed  Google Scholar 

  • Anchordoguy T, Carpenter JF, Loomis SH, Crowe JH (1988) Mechanisms of interaction of amino acids with phospholipid bilayers during freezing. Biochim Biophys Acta 946:299–306

    Article  CAS  PubMed  Google Scholar 

  • Beauchamp C, Fridovich I (1971) Superoxide dismutase: improved assays and assay applicable to acrylamide gels. Anal Biochem 44:276–287

    Article  CAS  PubMed  Google Scholar 

  • Belarmino MM, Mii M (2000) Agrobacterium-mediated genetic transformation of a Phalaenopsis orchid. Plant Cell Rep 19:435–442

    Article  CAS  Google Scholar 

  • Campos PS, Quartin V, Ramalho JC, Nunes MA (2003) Electrolyte leakage and lipid degradation account for cold sensitivity in leaves of Coffea sp. Plants J Plant Physiol 160:283–292

    Article  CAS  Google Scholar 

  • Chai ML, Xu CJ, Senthil KK, Kim JY, Kim DH (2002) Stable transformation of protocorm-like bodies in Phalaenopsis orchid mediated by Agrobacterium tumefaciens. Sci Hortic 96:213–224

    Article  CAS  Google Scholar 

  • Chan YL, Lin KH, Liao LJ, Chen WH, Chan MT (2005) Gene stacking in Phalaenopsis orchid enhances dual tolerance to pathogen attack. Transgenic Res 14:279–288

    Article  CAS  PubMed  Google Scholar 

  • Choi AM, Lee SB, Cho SH, Inhwan H, Cheol-Goo H, Suh MC (2008) Isolation and characterization of multiple abundant lipid transfer protein isoforms in developing sesame (Sesamum indicum L.) seeds. Plant Physiol Biochem 46:127–139

    Article  CAS  PubMed  Google Scholar 

  • Christie PJ, Alfenito MR, Walbot V (1994) Impact of low-temperature stress on general phenylpropanoid and anthocyanin pathways: enhancement of transcript abundance and anthocyanin pigmentation in maize seedlings. Planta 194:541–549

    Article  CAS  Google Scholar 

  • Crowe JH, Crowe LM, Carpenter JF, Rudolph AS, Wistrom CA, Spargo BJ, Anchordoguy TJ (1988) Interactions of sugars with membranes. Biochem Biophy Acta 947:367–384

    CAS  Google Scholar 

  • Eagles CF, Williams J, Louis DV (1993) Recovery after freezing effect of sugar concentration on cold acclimation in Avena sativa L. Lolium perenne L. and L. multiorum Lam. New Phytol 123:477–483

    Article  Google Scholar 

  • Estruch JJ, Carozzi NB, Desai N, Duck NB, Warren GW, Koziel MG (1997) Transgenic plants: an emerging approach to pest control. Nat Biotechnol 15:137–141

    Article  CAS  PubMed  Google Scholar 

  • Hughes MA, Dunn MA, Pearce RS, White AJ, Zhang L (1992) An abscisic-acid-responsive, low temperature barley gene has homology with a maize phospholipid transfer protein. Plant Cell Environ 15:861–865

    Article  CAS  Google Scholar 

  • Jung HW, Kim W, Hwang BK (2003) Three pathogen-inducible genes encoding lipid transfer protein from pepper are differentially activated by pathogens, abiotic, and environmental stresses. Plant Cell Environ 26:915–928

    Article  CAS  PubMed  Google Scholar 

  • Kirubakaran SI, Begum SM, Ulaganathan K, Sakthivel N (2008) Characterization of a new antifungal lipid transfer protein from wheat. Plant Physiol Biochem 46:918–927

    Article  CAS  PubMed  Google Scholar 

  • Kurodu H, Sagisaka S, Chiba K (1992) Collapse of peroxide-scavenging systems in apple flower-buds associated with freezing injury. Plant Cell Physiol 33:743–750

    Google Scholar 

  • Kurokawa T, Itagaki S, Yamaji T, Nakata C, Noda T, Hirano T (2006) Antioxidant activity of a novel extract from bamboo grass (AHSS) against ischemia–reperfusion injury in rat small intestine. Biol Pharm Bull 29:2301–2303

    Article  CAS  PubMed  Google Scholar 

  • Li CB (2004) Cloning of OsLTP1 gene from the Brazilian upland rice cv. IAPAR 9, its expression analysis and function identification. Dissertation of Institute of Genetics and Developmental Biology, Beijing, Chinese Academy of Sciences

  • Liau CH, You SJ, Prasad V, Hsiao HH, Lu JC, Yang NS, Chan MT (2003) Agrobacterium tumefaciens-mediated transformation of an Oncidium orchid. Plant Cell Rep 21:993–998

    Article  CAS  PubMed  Google Scholar 

  • Men S, Ming X, Wang Y, Liu R, Wei C, Li Y (2003) Genetic transformation of two species of orchid by biolistic bombardment. Plant Cell Rep 21:592–598

    CAS  PubMed  Google Scholar 

  • Mishiba K, Chin DP, Mii M (2005) Agrobacterium-mediated transformation of Phalaenopsis by targeting protocorms at an early stage after germination. Plant Cell Rep 24:297–303

    Article  CAS  PubMed  Google Scholar 

  • Offringa R, Lee F (1995) Isolation and characterization of plant genomic DNA sequences via (inverse) PCR amplification. In: Jones H (ed) Methods in molecular biology: plant gene transfer and expression protocols, vol 49. Humana Press, Totowa, pp 181–195

    Chapter  Google Scholar 

  • Ohkawa H, Ohishi N, Yagi K (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95:351–358

    Article  CAS  PubMed  Google Scholar 

  • Pearce RS, Dunn MA, Rixon JE, Harrison P, Hughes MA (1996) Expression of cold-inducible genes and frost hardiness in the crown meristem of young barley (Hordeum vulgare L. cv. Igri) plants grown in different environments. Plant Cell Environ 19:275–290

    Article  CAS  Google Scholar 

  • Polle A (1997) Defense against photooxidative damage in plants. In: Scandalios JG (ed) Oxidative stress and the molecular biology of antioxidant defenses. Cold Spring Harbour Laboratory Press, Plainview, pp 623–666

    Google Scholar 

  • Sagisaka S (1985) Injuries of cold acclimatized poplar twigs resulting from enzyme inactivation and substrate depression during frozen storage at ambient temperatures for a long period. Plant Cell Physiol 26:1135–1145

    CAS  Google Scholar 

  • Santarius KA (1992) Freezing of isolated thylakoid membranes in complex media. VIII. Differential cryoprotection by sucrose, proline and glycerol. Physiol Plant 84:87–93

    Article  CAS  Google Scholar 

  • Santoro MM, Liu Y, Khan SMA, Hou L-X, Bolen DW (1992) Increased thermal stability of proteins in the presence of naturally occurring osmolytes. Biochem 31:5278–5283

    Article  CAS  Google Scholar 

  • SAS Institute Inc (2001) SAS/START user’s guide version 8.2. SAS Institute, Cary

    Google Scholar 

  • Semiarti E, Indrianto A, Purwantoro A, Isminingsih S, Suseno N, Ishikawa T, Yoshioka Y, Machida Y, Machida C (2007) Agrobacterium-mediated transformation of the wild orchid species Phalaenopsis amabilis. Plant Biotechnol 24:265–272

    CAS  Google Scholar 

  • Shalaev EY, Steponkus PL (2001) Phase behavior and glass transition of 1, 2-dioleoylphosphaditylethanolamine (DOPE) dehydrated in the presence of sucrose. Biochem Biophys Acta Biomembr 1514:100–116

    Article  CAS  Google Scholar 

  • Shao HB, Liang ZS, Shao MA (2006) Osmotic regulation of 10 wheat (Triticum aestivum L.) genotypes at soil water deficits. Biointer 47:132–139

    Article  CAS  Google Scholar 

  • Shao HB, Chu LY, Lu ZH, Kang CM (2008) Primary antioxidant free radical scavenging and redox signaling pathways in higher plant cells. Int J Biol Sci 4:8–14

    CAS  Google Scholar 

  • Smirnoff N, Cumbes QJ (1989) Hydroxyl radical scavenging activity of compatible solutes. Phytochem 28:1057–1060

    Article  CAS  Google Scholar 

  • Srinivas V, Balasubramanian D (1995) Proline is a protein-compatible hydrotrope. Langmuir 11:2830–2833

    Article  CAS  Google Scholar 

  • Steponkus PL, Uemura M, Webb MS (1993) A contrast of the cryostability of the plasma membrane of winter rye and spring oat-two species that widely differ in their freezing tolerance and plasma membrane lipid composition. In: Steponkus PL (ed) Adv Low-Temp Biol, vol 2. Jai Press, London, pp 211–312

    Google Scholar 

  • Su V, Hsu BD (2003) Cloning and expression of a putative cytochrome P450 gene that influences the colour of Phalaenopsis flowers. Biotechnol Lett 25:1933–1939

    Article  CAS  PubMed  Google Scholar 

  • Trunova TI (1982) Mechanisms of winter wheat hardening at low temperature. In: Li PH, Sakai A (eds) Plant cold hardiness and freezing stress, vol 2. New York, Academic Press, pp 41–47

    Google Scholar 

  • Walker MA, McKersie BD (1993) Role of ascorbate-glutathione antioxidant system in chilling resistance of tomato. J Plant Physiol 141:234–239

    CAS  Google Scholar 

  • Wise R, Naylor A (1987) Chilling enhanced photooxidation. Evidence for the role of singlet oxygen and superoxide in the breakdown of pigments and endogenous antioxidants. Plant Physiol 83:278–282

    Article  CAS  PubMed  Google Scholar 

  • Xuan J, Liu J, Gao H, Hu H, Cheng X (2009) Evaluation of low-temperature tolerance of zoysia grass. Trop Grasslands 43:118–124

    Google Scholar 

Download references

Acknowledgments

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

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Authors and Affiliations

  1. College of Bioscience and Biotechnology, Yangzhou University, Yangzhou, 225009, China

    Xuyin Qin, Yang Liu, Shanjing Mao, Tianbi Li & Youping Wang

  2. Yangping Horticulture Co. Ltd, Yangzhou, 225008, China

    Hsinkan Wu

  3. Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China

    Chengcai Chu

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  1. Xuyin Qin
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  2. Yang Liu
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  3. Shanjing Mao
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  4. Tianbi Li
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  5. Hsinkan Wu
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  7. Youping Wang
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Correspondence to Youping Wang.

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Qin, X., Liu, Y., Mao, S. et al. Genetic transformation of lipid transfer protein encoding gene in Phalaenopsis amabilis to enhance cold resistance. Euphytica 177, 33–43 (2011). https://doi.org/10.1007/s10681-010-0246-4

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  • DOI: https://doi.org/10.1007/s10681-010-0246-4

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